Fused imidazo-piperidine jak inhibitor compound

ABSTRACT

The invention provides a compound of formula 1 
     
       
         
         
             
             
         
       
     
     or a pharmaceutically-acceptable salt thereof, that is useful as a JAK inhibitor. The invention also provides crystalline forms of the compound, pharmaceutical compositions comprising the compound, methods of using the compound to treat diseases amenable to a JAK inhibitor, and processes and intermediates useful for preparing the compound.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.62/492,574, filed on May 1, 2017, the disclosure of which isincorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION Field of the Invention

The invention is directed to a JAK kinase inhibitor compound useful forthe treatment of multiple diseases, particularly ocular, skin, andrespiratory diseases. The invention is also directed to crystallineforms of the compound, pharmaceutical compositions comprising such acompound, methods of using such a compound to treat diseases amenable totreatment with a JAK inhibitor, and processes and intermediates usefulfor preparing the compound.

State of the Art

Cytokines are intercellular signaling molecules which includechemokines, interferons, interleukins, lymphokines, and tumour necrosisfactor. Cytokines are critical for normal cell growth andimmunoregulation but also drive immune-mediated diseases and contributeto the growth of malignant cells. Elevated levels of many cytokines havebeen implicated in the pathology of a large number of diseases orconditions, particularly those diseases characterized by inflammation.Many of the cytokines implicated in disease act through signalingpathways dependent upon the Janus family of tyrosine kinases (JAKs),which signal through the Signal Transducer and Activator ofTranscription (STAT) family of transcription factors.

The JAK family comprises four members, JAK1, JAK2, JAK3, and tyrosinekinase 2 (TYK2). Binding of cytokine to a JAK-dependent cytokinereceptor induces receptor dimerization which results in phosphorylationof tyrosine residues on the JAK kinase, effecting JAK activation.Phosphorylated JAKs, in turn, bind and phosphorylate various STATproteins which dimerize, internalize in the cell nucleus and directlymodulate gene transcription, leading, among other effects, to thedownstream effects associated with inflammatory disease. The JAKsusually associate with cytokine receptors in pairs as homodimers orheterodimers. Specific cytokines are associated with specific JAKpairings. Each of the four members of the JAK family is implicated inthe signaling of at least one of the cytokines associated withinflammation.

Inflammation plays a prominent role in many ocular diseases, includinguveitis, diabetic retinopathy, diabetic macular edema, dry eye disease,age-related macular degeneration, retinal vein occlusion and atopickeratoconjunctivitis. Uveitis encompasses multiple intraocularinflammatory conditions and is often autoimmune, arising without a knowninfectious trigger. The condition is estimated to affect about 2 millionpatients in the US. In some patients, the chronic inflammationassociated with uveitis leads to tissue destruction, and it is the fifthleading cause of blindness in the US. Cytokines elevated in uveitispatients' eyes that signal through the JAK-STAT pathway include IL-2,IL-4, IL-4, 5, IL-6, IL-10, IL-23, and IFN-γ. (Horai and Caspi, JInterferon Cytokine Res, 2011, 31, 733-744; Ooi et al, Clinical Medicineand Research, 2006, 4, 294-309). Existing therapies for uveitis areoften suboptimal, and many patients are poorly controlled. Steroids,while often effective, are associated with cataracts and increasedintraocular pressure/glaucoma.

Diabetic retinopathy (DR) is caused by damage to the blood vessels inthe retina. It is the most common cause of vision loss among people withdiabetes. Angiogenic as well as inflammatory pathways play an importantrole in the disease. Often, DR will progress to diabetic macular edema(DME), the most frequent cause of visual loss in patients with diabetes.The condition is estimated to affect about 1.5 million patients in theUS alone, of whom about 20% have disease affecting both eyes. Cytokineswhich signal through the JAK-STAT pathway, such as IL-6, as well asother cytokines, such as IP-10 and MCP-1 (alternatively termed CCL2),whose production is driven in part by JAK-STAT pathway signaling, arebelieved to play a role in the inflammation associated with DR/DME(Abcouwer, J Clin Cell Immunol, 2013, Suppl 1, 1-12; Sohn et al.,American Journal of Opthalmology, 2011, 152, 686-694; Owen and Hartnett,Curr Diab Rep, 2013, 13, 476-480; Cheung et al, Molecular Vision, 2012,18, 830-837; Dong et al, Molecular Vision, 2013, 19, 1734-1746; Funatsuet al, Ophthalmology, 2009, 116, 73-79). The existing therapies for DMEare suboptimal: intravitreal anti-VEGF treatments are only effective ina fraction of patients and steroids are associated with cataracts andincreased intraocular pressure.

Dry eye disease (DED) is a multifactorial disorder that affectsapproximately 5 million patients in the US. Ocular surface inflammationis believed to play an important role in the development and propagationof this disease. Elevated levels of cytokines such as IL-1, IL-2, IL-4,IL-5, IL-6, and IFN-γ have been noted in the ocular fluids of patientswith DED. (Stevenson et al, Arch Ophthalmol, 2012, 130, 90-100), and thelevels often correlated with disease severity. Age-related maculardegeneration and atopic keratoconjunctivitis are also thought to beassociated with JAK-dependent cytokines.

Retinal vein occlusion (RVO) is a highly prevalent visually disablingdisease. Obstruction of retinal blood flow can lead to damage of theretinal vasculature, hemorrhage, and tissue ischemia. Although thecauses for RVO are multifactorial, both vascular as well as inflammatorymediators have been shown to be important (Deobhakta et al,International Journal of Inflammation, 2013, article ID 438412).Cytokines which signal through the JAK-STAT pathway, such as IL-6 andIL-13, as well as other cytokines, such as MCP-1, whose production isdriven in part by JAK-STAT pathway signaling, have been detected atelevated levels in ocular tissues of patients with RVO (Shchuko et al,Indian Journal of Ophthalmology, 2015, 63(12), 905-911). While manypatients with RVO are treated by photocoagulation, this is an inherentlydestructive therapy. Anti-VEGF agents are also used, but they are onlyeffective in a fraction of patients. Steroid medications that reduce thelevel of inflammation in the eye (Triamcinolone acetonide anddexamethasone implants) have also been shown to provide beneficialresults for patients with certain forms of RVO, but they have also beenshown to cause cataracts and increased intraocular pressure/glaucoma.

Atopic dermatitis (AD) is a common chronic inflammatory skin diseasethat affects an estimated 14 million people in the United States alone.It is estimated that AD affects 10-20% of children and 1-3% of adults indeveloped countries (Bao et al., JAK-STAT, 2013, 2, e24137) and theprevalence is increasing. Elevation of proinflammatory cytokines thatrely on the JAK-STAT pathway, in particular, IL-4, IL-5, IL-10, IL-13,and IFNγ, have been associated with AD (Bao et al., Leung et al., TheJournal of Clinical Investigation, 2004, 113, 651-657). In addition,upregulation of IL-31, another cytokine that signals through a JAKpairing, has been shown to have a role in the pruritis associated withthe chronic state of AD. (Sunkoly et al., Journal of Allergy andClinical Immunology, 2006, 117, 411-417)

Asthma is a chronic disease of the airways for which there are nopreventions or cures. The disease is characterized by inflammation,fibrosis, hyperresponsiveness, and remodeling of the airways, all ofwhich contribute to airflow limitation. An estimated 300 million peopleworldwide suffer from asthma and it is estimated that the number ofpeople with asthma will grow by more than 100 million by 2025. Althoughmost patients can achieve control of asthma symptoms with the use ofinhaled corticosteroids that may be combined with a leukotriene modifierand/or a long acting beta agonist, there remains a subset of patientswith severe asthma whose disease is not controlled by conventionaltherapies. Cytokines implicated in asthma inflammation which signalthrough the JAK-STAT pathway include IL-2, IL-3, IL-4, IL-5, IL-6, IL-9,IL-11, IL-13, IL-23, IL-31, IL-27, thymic stromal lymphopoietin (TSLP),interferon-γ (IFNγ) and granulocyte-macrophage colony-stimulating factor(GM-CSF). Inflammation of the airways is characteristic of otherrespiratory diseases in addition to asthma. Chronic obstructivepulmonary disease (COPD), cystic fibrosis (CF), pneumonitis,interstitial lung diseases (including idiopathic pulmonary fibrosis),acute lung injury, acute respiratory distress syndrome, bronchitis,emphysema, bronchiolitis obliterans, and sarcoidosis are alsorespiratory tract diseases in which the pathophysiology is believed tobe related to JAK-signaling cytokines.

Given the number of cytokines elevated in inflammatory diseases and thateach cytokine is associated with a particular JAK pairing, a chemicalinhibitor with pan-activity against all members of the JAK family couldhave broad utility for the treatment of ocular, skin, and respiratorydisease. The need remains for a potent pan-JAK inhibitor.

SUMMARY OF THE INVENTION

In one aspect, the invention provides a JAK inhibitor compound usefulfor the treatment of inflammatory disease.

In particular, in one aspect, the invention provides1-(2-(6-(2-ethyl-5-fluoro-4-hydroxyphenyl)-4-fluoro-1H-indazol-3-yl)-1,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridin-5-yl)-2-morpholinoethan-1-oneof the formula

hereinafter compound 1, or a pharmaceutically-acceptable salt thereof.

The invention also provides crystalline forms of the compound, Form 1and Form 2.

The invention also provides a pharmaceutical composition comprisingcompound 1, or a pharmaceutically acceptable salt thereof, and apharmaceutically-acceptable carrier.

In one aspect, the invention provides a method of treating an oculardisease in a mammal, the method comprising administering to the mammalcompound 1, or a pharmaceutical composition of the invention. In oneaspect the ocular disease is uveitis, diabetic retinopathy, diabeticmacular edema, dry eye disease, age-related macular degeneration,retinal vein occlusion and atopic keratoconjunctivitis. In particular,the ocular disease is diabetic macular edema or uveitis.

In yet another method aspect, the invention provides a method oftreating an inflammatory disease of the skin, in particular atopicdermatitis, the method comprising applying compound 1, or apharmaceutical composition of the invention to the skin of the mammal.

In a further aspect, the invention provides a method of treating arespiratory disease in a mammal, the method comprising administeringcompound 1, or a pharmaceutically-acceptable salt thereof or apharmaceutical composition of the invention to the mammal.

In separate and distinct aspects, the invention also provides syntheticprocesses and intermediates described herein, which are useful forpreparing compound 1.

The invention also provides compound 1 as described herein for use inmedical therapy, as well as the use of the compound of the invention inthe manufacture of a formulation or medicament for treating oculardisease, skin disease, or respiratory disease in a mammal.

BRIEF DESCRIPTION OF THE DRAWINGS

Various aspects of the present invention are illustrated by reference tothe accompanying drawings.

FIG. 1 shows a powder x-ray diffraction (PXRD) pattern of crystallineForm 2 of compound 1 (hereinafter Form 2).

FIG. 2 shows a differential scanning calorimetry (DSC) thermogram ofcrystalline Form 2.

FIG. 3 shows a thermal gravimetric analysis (TGA) plot of crystallineForm 2.

FIG. 4 shows a dynamic moisture sorption (DMS) isotherm of crystallineForm 2 observed at a temperature of about 25° C.

FIG. 5 shows a polarized light microscope image of Form 2.

FIG. 6 shows a powder x-ray diffraction (PXRD) pattern of crystallineForm 1 of compound 1 (hereinafter Form 1).

FIG. 7 shows a differential scanning calorimetry (DSC) thermogram ofcrystalline Form 1.

FIG. 8 shows a thermal gravimetric analysis (TGA) plot of crystallineForm 1.

FIG. 9 shows a dynamic moisture sorption (DMS) isotherm of crystallineForm 1 observed at a temperature of about 25° C.

FIG. 10 shows a polarized light microscopy image of Form 1.

DETAILED DESCRIPTION OF THE INVENTION

Chemical structures are named herein according to IUPAC conventions asimplemented in ChemDraw software (PerkinElmer, Inc., Cambridge, Mass.).

Furthermore, the imidazo portion of the tetrahydroimidazopyridine moietyin the structure of the present compound exists in tautomeric forms. Thecompound could equivalently be represented as

According to the IUPAC convention, these representations give rise todifferent numbering of the atoms of the tetrahydroimidazopyridineportion. Accordingly this structure is designated1-(2-(6-(2-ethyl-5-fluoro-4-hydroxyphenyl)-4-fluoro-1H-indazol-3-yl)-3,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridin-5-yl)-2-morpholinoethan-1-one.It can also be designated1-(2-(6-(2-ethyl-5-fluoro-4-hydroxyphenyl)-4-fluoro-1H-indazol-3-yl)-1,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridin-5-yl)-2-morpholinoethan-1-one.It will be understood that although structures are shown, or named, in aparticular form, the invention also includes the tautomer thereof.

The compounds of the invention contain several basic groups andtherefore, the compounds can exist as the free base or in various saltforms, such as a mono-protonated salt form, a di-protonated salt form,or mixtures thereof. All such forms are included within the scope ofthis invention, unless otherwise indicated.

This invention also includes isotopically-labeled compounds of formula1, i.e., compounds of formula 1 where an atom has been replaced orenriched with an atom having the same atomic number but an atomic massdifferent from the atomic mass that predominates in nature. Examples ofisotopes that may be incorporated into a compound of formula 1 include,but are not limited to, ²H, ³H, ¹¹C, ¹³C, ¹⁴C, ¹³N, ¹⁵N, ¹⁵O, ¹⁷O, ¹⁸O,and ¹⁸F. Of particular interest are compounds of formula 1 enriched intritium or carbon-14, which compounds can be used, for example, intissue distribution studies. Also of particular interest are compoundsof formula 1 enriched in deuterium especially at a site of metabolism,which compounds are expected to have greater metabolic stability.

Additionally of particular interest are compounds of formula 1 enrichedin a positron emitting isotope, such as ¹¹C, ¹⁸F, ¹⁵O and ¹³N, whichcompounds can be used, for example, in Positron Emission Tomography(PET) studies.

Definitions

When describing this invention including its various aspects andembodiments, the follow

The term “therapeutically effective amount” means an amount sufficientto effect treatment when administered to a patient in need of treatment.

The term “treating” or “treatment” means preventing, ameliorating orsuppressing the medical condition, disease or disorder being treated(e.g., a respiratory disease) in a patient (particularly a human); oralleviating the symptoms of the medical condition, disease or disorder.

The term “pharmaceutically acceptable salt” means a salt that isacceptable for administration to a patient or a mammal, such as a human(e.g., salts having acceptable mammalian safety for a given dosageregime). Representative pharmaceutically acceptable salts include saltsof acetic, ascorbic, benzenesulfonic, benzoic, camphorsulfonic, citric,ethanesulfonic, edisylic, fumaric, gentisic, gluconic, glucoronic,glutamic, hippuric, hydrobromic, hydrochloric, isethionic, lactic,lactobionic, maleic, malic, mandelic, methanesulfonic, mucic,naphthalenesulfonic, naphthalene-1,5-disulfonic,naphthalene-2,6-disulfonic, nicotinic, nitric, orotic, pamoic,pantothenic, phosphoric, succinic, sulfuric, tartaric, p-toluenesulfonicand xinafoic acid, and the like.

The term “salt thereof” means a compound formed when the hydrogen of anacid is replaced by a cation, such as a metal cation or an organiccation and the like. For example, the cation can be a protonated form ofa compound of formula 1, i.e. a form where one or more amino groups havebeen protonated by an acid. Typically, the salt is a pharmaceuticallyacceptable salt, although this is not required for salts of intermediatecompounds that are not intended for administration to a patient.

The term “amino-protecting group” means a protecting group suitable forpreventing undesired reactions at an amino nitrogen. Representativeamino-protecting groups include, but are not limited to, formyl; acylgroups, for example alkanoyl groups, such as acetyl andtri-fluoroacetyl; alkoxycarbonyl groups, such as tert butoxycarbonyl(Boc); arylmethoxycarbonyl groups, such as benzyloxycarbonyl (Cbz) and9-fluorenylmethoxycarbonyl (Fmoc); arylmethyl groups, such as benzyl(Bn), trityl (Tr), and 1,1-di-(4′-methoxyphenyl)methyl; silyl groups,such as trimethylsilyl (TMS), tert-butyldimethylsilyl (TBDMS),[2-(trimethylsilyl)ethoxy]methyl (SEM); and the like.

The term “hydroxy-protecting group” means a protecting group suitablefor preventing undesired reactions at a hydroxy group. Representativehydroxy-protecting groups include, but are not limited to, alkyl groups,such as methyl, ethyl, and tert-butyl; acyl groups, for example alkanoylgroups, such as acetyl; arylmethyl groups, such as benzyl (Bn),p-methoxybenzyl (PMB), 9-fluorenylmethyl (Fm), and diphenylmethyl(benzhydryl, DPM); silyl groups, such as trimethylsilyl (TMS) andtert-butyldimethylsilyl (TBS); and the like.

Numerous protecting groups, and their introduction and removal, aredescribed in T. W. Greene and P. G. M. Wuts, Protecting Groups inOrganic Synthesis, Third Edition, Wiley, New York

General Synthetic Procedures

Compound 1, and intermediates thereof, can be prepared according to thefollowing general methods and procedures using commercially-available orroutinely-prepared starting materials and reagents. Additionally,compounds having an acidic or basic atom or functional group may be usedor may be produced as a salt unless otherwise indicated (in some cases,the use of a salt in a particular reaction will require conversion ofthe salt to a non-salt form, e.g., a free base, using routine proceduresbefore conducting the reaction).

Although a particular embodiment of the present invention may be shownor described in the following procedures, those skilled in the art willrecognize that other embodiments or aspects of the present invention canalso be prepared using such procedures or by using other methods,reagents, and starting materials know to those skilled in the art. Inparticular, it will be appreciated that compound 1 may be prepared by avariety of process routes in which reactants are combined in differentorders to provide different intermediates en route to producing finalproducts.

The preparation of1-(2-(6-(2-ethyl-5-fluoro-4-hydroxyphenyl)-4-fluoro-1H-indazol-3-yl)-1,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridin-5-yl)-2-morpholinoethan-1-one(compound 1) is described in detail in the appended examples. Key stepsare summarized in Scheme 1

where reagent 7 is 2,5-dioxopyrrolidin-1-yl 2-morpholinoacetate, i.e.the variable R^(A) represents the activating agent2,5-dioxopyrrolidinyl, as described in Example 1. Alternatively,morpholin-4-yl acetic acid, i.e. R^(A) represents hydrogen, is used asreagent 7, under typical amide bond formation conditions, as describedin Example 4.

Intermediate 3 may be prepared as described in Preparations 1 and 3below. An alternative method of preparation of the key protectedintermediate 5 is illustrated in Scheme 2.

The bromoindazole aldehyde 8 may be reacted with the benzyl protectedimine compound 2 to provide intermediate 9. The reaction is typicallyconducted in the presence of sodium bisulfite, at a temperature ofbetween about 130° C. and about 140° C. for between about 1 and about 6hours or until the reaction is substantially complete. Compound 9 isreduced using a reducing agent such as sodium borohydride to providecompound 10, which is combined with protected phenyltrifluoroborate 11under typical Suzuki-Miyaura coupling conditions to provide intermediate5. The reaction is typically conducted at elevated temperature in thepresence of a palladium catalyst. The Suzuki partner 11, shown in Scheme2 as the trifluoroborate potassium salt can be prepared by reacting thecorresponding boronate (Intermediate 1-5 in Preparation 1 below) withpotassium hydrogen difluoride to provide intermediate 11. Alternatively,the boronate intermediate can be used in place of the trifluoroborate11.

Accordingly, in a method aspect, the invention provides a process ofpreparing a compound of formula 1 or a pharmaceutically acceptable saltthereof, the process comprising reacting a compound of formula 6 with acompound of formula 7, as illustrated in Scheme 1 to provide a compoundof formula 1 or a pharmaceutically acceptable salt thereof.

In an additional method aspect, the invention provides a compound offormula 5 and a compound of formula 6, useful in preparing a compound offormula 1.

Crystalline Forms

In another aspect, the invention provides1-(2-(6-(2-Ethyl-5-fluoro-4-hydroxyphenyl)-4-fluoro-1H-indazol-3-yl)-1,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridin-5-yl)-2-morpholinoethan-1-one(1) in crystalline form.

Form 1

Crystalline Form 1 of the invention is a crystalline free form ofcompound 1. In one aspect, Form 1 is characterized by a powder X-raydiffraction (PXRD) pattern having significant diffraction peaks, amongother peaks, at 2θ values of 8.16±0.20, 8.97±0.20, 15.29±0.20,16.70±0.20, 18.00±0.20, and 20.18±0.20. Form 1 may be furthercharacterized by a PXRD pattern having two or more additionaldiffraction peaks, including three or more and four or more additionaldiffraction peaks at 2θ values selected from 7.69±0.20, 10.66±0.20,11.46±0.20, 11.91±0.20, 15.80±0.20, 17.02±0.20, 18.83±0.20, 22.39±0.20,22.98±0.20, 24.89±0.20, and 26.54±0.20. In another aspect, Form 1 ischaracterized by a PXRD pattern having three, four, five, or sixdiffraction peaks at 2θ values selected from 8.16±0.20, 8.97±0.20,15.29±0.20, 16.70±0.20, 18.00±0.20, and 20.18±0.20.

As is well known in the field of powder X-ray diffraction, peakpositions of PXRD pattern are relatively less sensitive to experimentaldetails, such as details of sample preparation and instrument geometry,than are the relative peak heights. Thus, in one aspect, the crystallineForm 1 is characterized by a powder x-ray diffraction pattern in whichthe peak positions are substantially in accordance with those shown inFIG. 6.

In another aspect, crystalline Form 1 is characterized by its behaviorwhen exposed to high temperature. As demonstrated in FIG. 7, thedifferential scanning calorimetry (DSC) trace recorded at a heating rateof 10° C. per minute exhibits a peak in endothermic heat flow,identified as a melt transition, in the range of about 210° C. to about234° C., or in the range of between about 215° C. to about 229° C., orin the range of between about 220° C. to about 224° C. The crystallineForm 1 is characterized by a differential scanning calorimetry tracerecorded at a heating rate of 10° C. per minute which shows a maximum inendothermic heat flow with a peak at about 221.7° C. The thermalgravimetric analysis (TGA) trace of FIG. 8 shows no significant weightloss at temperatures below the onset of decomposition at about 293° C.

A representative dynamic moisture sorption (DMS) trace for the Form 1crystalline free form of the invention is shown in FIG. 9. CrystallineForm 1 demonstrated a small hysteresis between two cycles of sorptionand desorption. Form 1 demonstrated about 0.99% weight gain in thehumidity range of 5% to 70% relative humidity and about 1.32% weightgain in the humidity range of 5% to 90% relative humidity at roomtemperature, as shown in FIG. 9. Form 1 is considered to beslightly-hygroscopic.

Crystalline Form 1 has been shown to be stable upon exposure to elevatedtemperature and humidity. After 36 weeks at accelerated conditions of40° C. and 75% relative humidity, no statistically significant changesin chemical purity were observed.

Form 1 may be prepared by dissolving compound 1 in ethanol upon heating,followed by addition of acetronitrile, wherein the ratio of acetonitrileto ethanol is about 1:1, or from about 1:3 to 3:1. The resulting mixtureis then warmed, followed by stirring at a temperature of between about20° C. and about 25° C. for between about 4 hours and about 30 hours, orfor about 16 hours. The solid is then filtered and dried to provide Form1.

Form 1 may also be prepared by mixing compound 1 with ethanol andstirring the mixture at a temperature of between about 50 and about 80°C. for about 2 to 30 minutes, or about 10 minutes, followed by slowaddition of acetonitrile at a temperature of between about 50 and about80° C., wherein the ratio in volume of acetonitrile to ethanol is fromabout 3:1 to 1:1 or about 1.5:1. Seeds of Form 1 may be added and thereaction mixture stirred at a temperature of between about 20° C. andabout 25° C. for between about 4 hours and about 30 hours, or for about18 hours. The solid is then filtered and dried to provide form 1.

Form 2

Crystalline Form 2 of the invention is a crystalline free form ofcompound 1. In one aspect, Form 2 is characterized by a powder X-raydiffraction (PXRD) pattern having significant diffraction peaks, amongother peaks, at 2θ values of 10.61±0.20, 11.84±0.20, 14.94±0.20,18.26±0.20, and 19.06±0.20. Form 2 may be further characterized by aPXRD pattern having additional diffraction peaks at 2θ values of13.32±0.20, 17.69±0.20, and 21.10±0.20. Form 2 may be furthercharacterized by a PXRD pattern having two or more additionaldiffraction peaks, including three or more and four or more additionaldiffraction peaks at 2θ values selected from 10.85±0.20, 16.14±0.20,16.35±0.20, 18.43±0.20, 19.20±0.20, 19.49±0.20, 20.72±0.20, 21.94±0.20,22.64±0.20, 23.64±0.20, 25.19±0.20, and 28.08±0.20.

In another aspect, Form 2 is characterized by a PXRD pattern havingthree, four, five, or six diffraction peaks at 2θ values selected from10.61±0.20, 11.84±0.20, 13.32±0.20, 14.94±0.20, 17.69±0.20, 18.26±0.20,19.06±0.20 and 21.10±0.20.

As is well known in the field of powder X-ray diffraction, peakpositions of PXRD pattern are relatively less sensitive to experimentaldetails, such as details of sample preparation and instrument geometry,than are the relative peak heights. Thus, in one aspect, the crystallineForm 2 is characterized by a powder x-ray diffraction pattern in whichthe peak positions are substantially in accordance with those shown inFIG. 1.

The structure of crystalline Form 2 has been further characterized bysingle crystal x-ray diffraction analysis. The crystals belong to anorthorhombic crystal system and Pbca space group. The unit celldimensions are: a=9.7245(11) Å, b=16.8197(14) Å, c=32.604(4) Å, α=900,β=90°, γ=900, volume=5332.8(10) ∈³. The calculated density is 1.302g/cm³. The crystals contain eight molecules per unit cell. The structureconfirms that the crystals do not contain water or other solventmolecules and the molecular structure is consistent with the structureof the compound of Example 1 as depicted herein. Powder X-raydiffraction peaks predicted from the derived atomic positions are ingood agreement with observed results.

In another aspect, crystalline Form 2 is characterized by its behaviorwhen exposed to high temperature. As demonstrated in FIG. 2, thedifferential scanning calorimetry (DSC) trace recorded at a heating rateof 10° C. per minute exhibits a peak in endothermic heat flow,identified as a melt transition, in the range of about 268° C. to about277° C., or in the range of between about 270° C. to about 275° C., orin the range of between about 271° C. to about 274° C. The crystallineForm 2 is characterized by a differential scanning calorimetry tracerecorded at a heating rate of 10° C. per minute which shows a maximum inendothermic heat flow with a peak at about 272.6±2° C.

The thermal gravimetric analysis (TGA) trace of FIG. 3 shows nosignificant weight loss at temperatures below the onset of decompositionat about 269° C.

A representative dynamic moisture sorption (DMS) trace for the Form 2crystalline free form of the invention is shown in FIG. 4. CrystallineForm 2 showed no hysteresis between two cycles of sorption anddesorption and demonstrated an exceptionally small propensity forhygroscopicity. Form 2 demonstrated about 0.18% weight gain in thehumidity range of 5% to 90% relative humidity and about 0.12% weightgain in the humidity range of 5% to 70% relative humidity at roomtemperature, as shown in FIG. 4. Form 2 is considered to benon-hygroscopic.

Form 2 can be prepared by dissolving compound 1 of example 1 in DMSO(for example, at a ratio of 1 g of compound 1 for 1 to 3 mL, or about 2mL of DMSO) at a temperature between about 45 and 75° C., or at about60° C., followed by addition of methanol, wherein the ratio in volume ofmethanol to DMSO is from about 1:4 to about 1:1, or about 1:2. Thehomogenous mixture is then added dropwise to a premixed solution ofmethanol and water (wherein the ratio of methanol to DMSO is between 1.5and 3 to 1), at a temperature between about 60 and about 90° C., orabout 75° C., wherein the ratio in volume of the premixed solution ofmethanol to water is from about 0.5:1 to about 1:2, or about 1:0.9. Themixture is then allowed to stir at a temperature between about 60 andabout 90° C., or about 75° C. for about 30 minutes to about 2 hours, orabout 1 hour. Water can then be slowly added at a temperature betweenabout 60 and about 90° C., or about 75° C., wherein the ratio in volumeof water to methanol is between 2 and 4. The resulting slurry is thenslowly cooled down to room temperature (typically, a temperature betweenabout 20 and about 25° C.), typically over about 2 to about 12 hours orabout 6 hours. The slurry is then held at room temperature and thenfiltered and washed with a mixture of water and methanol at about 50 toabout 90%, or about 70% water, to provide Form 2.

In one aspect, the invention provides a method of preparing crystallineForm 2 comprising:

-   -   (a) forming an homogenous mixture of        1-(2-(6-(2-ethyl-5-fluoro-4-hydroxyphenyl)-4-fluoro-1H-indazol-3-yl)-1,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridin-5-yl)-2-morpholinoethan-1-one        in a polar aprotic solvent, or in a polar water-miscible        solvent, or in a mixture of a polar aprotic solvent and a polar        water-miscible solvent, at a temperature between 45 and 75° C.;    -   (b) adding the homogenous mixture to a mixture of a water        miscible solvent and water at a temperature between 60 and        90° C. to give a second mixture;    -   (c) slowly adding water to the second mixture at a temperature        between 60 and 90° C. to form a slurry; and    -   (d) isolating the crystalline form from the slurry.

In some aspects, the polar aprotic solvent of step (a) is selected fromthe group consisting of DMSO, DMF, NMP, DMAc, and nitromethane, thepolar water-miscible solvent of step (a) is selected from the groupconsisting of acetonitrile, acetone, methanol, ethanol, and THF, and thewater miscible solvent of step (b) is selected from the group consistingof acetonitrile, acetone, methanol, ethanol, n-propanol, isopropanol,n-butanol, THF, DMSO, DMF, NMP, DMAc, and nitromethane. In some aspects,the polar aprotic solvent of step (a) is DMSO, the polar water-misciblesolvent of step (a) is methanol, and the water miscible solvent of step(b) is methanol.

In some aspects, the slurry obtained in step (c) is cooled down to atemperature between about 20 and 25° C. before step (d).

Alternatively, Form 2 can be formed by stirring the compound 1 obtainedin example 1 in a mixture of a polar water miscible solvent and water,at a temperature between 60 and 90° C. In some aspects, the ratio ofsolvent to water is about 1:1, or from 2:1 to 0.5:1. In some aspects,the polar water miscible solvent is selected from the group consistingof acetonitrile, acetone, methanol, ethanol, n-propanol, isopropanol,n-butanol, THF, DMSO, DMF, NMP, DMAc, and nitromethane.

Pharmaceutical Compositions

Compound 1, and pharmaceutically-acceptable salts thereof are typicallyused in the form of a pharmaceutical composition or formulation. Suchpharmaceutical compositions may advantageously be administered to apatient by any acceptable route of administration including, but notlimited to, oral, inhalation, optical injection, topical (includingtransdermal), rectal, nasal, and parenteral modes of administration.

Accordingly, in one of its compositions aspects, the invention isdirected to a pharmaceutical composition comprising apharmaceutically-acceptable carrier or excipient and compound 1, where,as defined above, “compound 1” means compound 1 or apharmaceutically-acceptable salt thereof. Optionally, suchpharmaceutical compositions may contain other therapeutic and/orformulating agents if desired. When discussing compositions and usesthereof, compound 1 may also be referred to herein as the “activeagent”.

In some aspects, the disclosure provides a pharmaceutical compositioncomprising compound 1, or a pharmaceutically acceptable salt thereof, orForm 1 or Form 2 and a pharmaceutically-acceptable carrier. In someaspects, the pharmaceutical composition is suitable for application tothe eye. In some aspects, the composition is suitable for injection intothe eye. In some aspects, the composition is suitable for intravitrealinjection. In some aspects, the composition is a suspension. In someaspects, the composition is a crystalline suspension. In some aspects,the composition is a suspension of Form 1 or Form 2.

The pharmaceutical compositions of the invention typically contain atherapeutically effective amount of compound 1. Those skilled in the artwill recognize, however, that a pharmaceutical composition may containmore than a therapeutically effective amount, i.e., bulk compositions,or less than a therapeutically effective amount, i.e., individual unitdoses designed for multiple administration to achieve a therapeuticallyeffective amount.

Typically, such pharmaceutical compositions will contain from about 0.01to about 95% by weight of the active agent; including, for example, fromabout 0.05 to about 30% by weight; and from about 0.1% to about 10% byweight of the active agent.

Any conventional carrier or excipient may be used in the pharmaceuticalcompositions of the invention. The choice of a particular carrier orexcipient, or combinations of carriers or excipients, will depend on themode of administration being used to treat a particular patient or typeof medical condition or disease state. In this regard, the preparationof a suitable pharmaceutical composition for a particular mode ofadministration is well within the scope of those skilled in thepharmaceutical arts.

Additionally, the carriers or excipients used in the pharmaceuticalcompositions of this invention are commercially-available. By way offurther illustration, conventional formulation techniques are describedin Remington: The Science and Practice of Pharmacy, 20th Edition,Lippincott Williams & White, Baltimore, Md. (2000); and H. C. Ansel etal., Pharmaceutical Dosage Forms and Drug Delivery Systems, 7th Edition,Lippincott Williams & White, Baltimore, Md. (1999).

Representative examples of materials which can serve as pharmaceuticallyacceptable carriers include, but are not limited to, the following:sugars, such as lactose, glucose and sucrose; starches, such as cornstarch and potato starch; cellulose, such as microcrystalline cellulose,and its derivatives, such as sodium carboxymethyl cellulose, ethylcellulose and cellulose acetate; powdered tragacanth; malt; gelatin;talc; excipients, such as cocoa butter and suppository waxes; oils, suchas peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil,corn oil and soybean oil; glycols, such as propylene glycol; polyols,such as glycerin, sorbitol, mannitol and polyethylene glycol; esters,such as ethyl oleate and ethyl laurate; agar; buffering agents, such asmagnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-freewater; isotonic saline; Ringer's solution; ethyl alcohol; phosphatebuffer solutions; and other non-toxic compatible substances employed inpharmaceutical compositions.

Pharmaceutical compositions are typically prepared by thoroughly andintimately mixing or blending the active agent with apharmaceutically-acceptable carrier and one or more optionalingredients. The resulting uniformly blended mixture can then be shapedor loaded into tablets, capsules, pills and the like using conventionalprocedures and equipment.

The pharmaceutical compositions of the invention are preferably packagedin a unit dosage form. The term “unit dosage form” refers to aphysically discrete unit suitable for dosing a patient, i.e., each unitcontaining a predetermined quantity of active agent calculated toproduce the desired therapeutic effect either alone or in combinationwith one or more additional units. For example, such unit dosage formsmay be capsules, tablets, pills, and the like, or unit packages suitablefor ocular or parenteral administration.

In one embodiment, the pharmaceutical compositions of the invention aresuitable for oral administration. Suitable pharmaceutical compositionsfor oral administration may be in the form of capsules, tablets, pills,lozenges, cachets, dragees, powders, granules; or as a solution or asuspension in an aqueous or non-aqueous liquid; or as an oil-in-water orwater-in-oil liquid emulsion; or as an elixir or syrup; and the like;each containing a predetermined amount of compound 1 as an activeingredient.

When intended for oral administration in a solid dosage form (i.e., ascapsules, tablets, pills and the like), the pharmaceutical compositionsof the invention will typically comprise the active agent and one ormore pharmaceutically-acceptable carriers. Optionally, such solid dosageforms may comprise: fillers or extenders, such as starches,microcrystalline cellulose, lactose, dicalcium phosphate, sucrose,glucose, mannitol, and/or silicic acid; binders, such ascarboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone,sucrose and/or acacia; humectants, such as glycerol; disintegratingagents, such as crosscarmellose sodium, agar-agar, calcium carbonate,potato or tapioca starch, alginic acid, certain silicates, and/or sodiumcarbonate; solution retarding agents, such as paraffin; absorptionaccelerators, such as quaternary ammonium compounds; wetting agents,such as cetyl alcohol and/or glycerol monostearate; absorbents, such askaolin and/or bentonite clay; lubricants, such as talc, calciumstearate, magnesium stearate, solid polyethylene glycols, sodium laurylsulfate, and/or mixtures thereof; coloring agents; and buffering agents.

Release agents, wetting agents, coating agents, sweetening, flavoringand perfuming agents, preservatives and antioxidants can also be presentin the pharmaceutical compositions of the invention. Examples ofpharmaceutically-acceptable antioxidants include: water-solubleantioxidants, such as ascorbic acid, cysteine hydrochloride, sodiumbisulfate, sodium metabisulfate, sodium sulfite and the like;oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole, butylated hydroxytoluene, lecithin, propyl gallate,alpha-tocopherol, and the like; and metal-chelating agents, such ascitric acid, ethylenediamine tetraacetic acid, sorbitol, tartaric acid,phosphoric acid, and the like. Coating agents for tablets, capsules,pills and like, include those used for enteric coatings, such ascellulose acetate phthalate, polyvinyl acetate phthalate, hydroxypropylmethylcellulose phthalate, hydroxypropyl methylcellulose, methacrylicacid, methacrylic acid ester copolymers, cellulose acetate trimellitate,carboxymethyl ethyl cellulose, hydroxypropyl methyl cellulose acetatesuccinate, and the like.

Pharmaceutical compositions of the invention may also be formulated toprovide slow or controlled release of the active agent using, by way ofexample, hydroxypropyl methyl cellulose in varying proportions; or otherpolymer matrices, liposomes and/or microspheres. In addition, thepharmaceutical compositions of the invention may optionally containopacifying agents and may be formulated so that they release the activeingredient only, or preferentially, in a certain portion of thegastrointestinal tract, optionally, in a delayed manner. Examples ofembedding compositions which can be used include polymeric substancesand waxes. The active agent can also be in micro-encapsulated form, ifappropriate, with one or more of the above-described excipients.

Suitable liquid dosage forms for oral administration include, by way ofillustration, pharmaceutically-acceptable emulsions, microemulsions,solutions, suspensions, syrups and elixirs. Liquid dosage formstypically comprise the active agent and an inert diluent, such as, forexample, water or other solvents, solubilizing agents and emulsifiers,such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethylacetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyleneglycol, oils (esp., cottonseed, groundnut, corn, germ, olive, castor andsesame oils), oleic acid, glycerol, tetrahydrofuryl alcohol,polyethylene glycols and fatty acid esters of sorbitan, and mixturesthereof. Alternatively, certain liquid formulations can be converted,for example, by spray drying, to a powder, which is used to preparesolid dosage forms by conventional procedures.

Suspensions, in addition to the active ingredient, may containsuspending agents such as, for example, ethoxylated isostearyl alcohols,polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth,and mixtures thereof.

Compound 1 can also be administered parenterally (e.g. by intravenous,subcutaneous, intramuscular or intraperitoneal injection). Forparenteral administration, the active agent is typically admixed with asuitable vehicle for parenteral administration including, by way ofexample, sterile aqueous solutions, saline, low molecular weightalcohols such as propylene glycol, polyethylene glycol, vegetable oils,gelatin, fatty acid esters such as ethyl oleate, and the like.Parenteral formulations may also contain one or more anti-oxidants,solubilizers, stabilizers, preservatives, wetting agents, emulsifiers,buffering agents, or dispersing agents. These formulations may berendered sterile by use of a sterile injectable medium, a sterilizingagent, filtration, irradiation, or heat.

Compound 1 may also be formulated as a sterile aqueous suspension orsolution for ocular injection. Useful excipients that may be included insuch an aqueous formulation include polysorbate 80, cellulose polymerssuch as carboxymethylcellulose, hydroxypropyl methylcellulose,methylcellulose, potassium chloride, calcium chloride, sodium chloride,magnesium chloride, sodium acetate, sodium citrate, histidine,α-a-trehalose dihydrate, sucrose, polysorbate 20,hydroxypropyl-3-cyclodextrin, benzalkonium chloride, Amberlite IRP-69,polyoxyethylene glycol ethers (lauryl, stearyl and oleyl),ethylenediaminetetra acetic acid sodium salt, sodium taurocholate,saponins and cremophor EL, polycarbophil-cysteine, Xanthan gum, Gellangum, hyaluronic acid, liposomes, and sodium phosphate. Permeabilityenhancers, surfactants, bile acids, cyclodextrins such as2-hydroxypropyl-β-cyclodextrin, and chelating agents may be included inthe formulation. Cylindrical oligonucleotides with a hydrophilic outersurface and a lipophilic inner surface that have the ability of formingcomplexes with an active agent may also be included in the formulation.Benzyl alcohol may serve as a preservative and sodium chloride may beincluded to adjust tonicity. In addition, hydrochloric acid and/orsodium hydroxide may be added to the solution for pH adjustment. Aqueousformulations for ocular injection may be prepared as preservative-free.

The ocular formulation may allow sustained release of the activeingredient to the eye. The ocular formulation may be formulated as anemulsion (oil in water or water in oil), a suspension, or an ointment.The suspension formulation may contain compound 1, or a pharmaceuticallyacceptable salt thereof, as a crystalline form, for example Form 1 orForm 2, or in an amorphous state.

Compound 1 may also be formulated to be suitable for eye drop dosing oras an intravitreal implant. The implant may allow delivering constanttherapeutic levels of drug. Reservoir implants are typically made with apelleted drug core surrounded by nonreactive substances such as silicon,ethylene vinyl acetate (EVA), or polyvinyl alcohol (PVA); these implantsare nonbiodegradable and can deliver continuous amounts of a drug formonths to year. Matrix implants may also be used. They are typicallyused to deliver a loading dose followed by tapering doses of the drugduring a 1-day to 6-month time period. They are most commonly made fromthe copolymers poly-lactic-acid (PLA) and/or poly-lactic-glycolic acid(PLGA), which degrade to water and carbon dioxide. Iontophoresis mayalso be used. It is a noninvasive technique in which a small electriccurrent is applied to enhance ionized drug penetration into tissue.

Encapsulated cell technology (ECT), which is a cell-based deliverysystem may also be used to deliver the therapeutic agent to the eye.Typically, genetically modified cells are packaged in a hollow tube ofsemipermeable membrane, which prevents immune-cell entry and allowsnutrients and therapeutic molecules to diffuse freely across themembrane. Two ends of the polymer section are sealed, and a titaniumloop is placed on the anchoring end, which is implanted at the parsplana and anchored to the sclera.

Compound 1 may be formulated into any form allowing delivery to the backof the eye. Examples of modes of delivery are known in the literature(Kuno et al, Polymers, 2011, 3, 193-221, del Amo et al, Drug DiscoveryToday, 2008, 13, 135-143, Short, Toxicologic Pathology, 2008, 36,49-62). Such modes of delivery include but are not limited tosuprachoroidal delivery which allows delivery to the choroid and retinathrough the suprachoroidal space, sub-Tenon delivery, peri-oculardelivery, contact lenses, punctal plugs, and scleral plugs. Compound 1may also be delivered by periocular, suprascleral, retrobulbar,peribulbar, or subconjunctival injection.

Compound 1 may be delivered as an emulsion, polymeric micro ornanospheres, liposomes, micro or nanoparticles, microspheres, micelles,or dendrimers. Biodegradable and biocompatible polymers, such aspolyactide and PLGA can be used. Compound 1 may be encapsulated.

In addition, compound 1 may be formulated for topical administration tothe skin as an ointment or cream. Ointment formulations are semisolidpreparations having a base of an oily or greasy material that istypically clear. Suitable oily materials for use in ointmentformulations include petrolatum (petroleum jelly), beeswax, cocoabutter, shea butter, and cetyl alcohol. Ointments may optionallyadditionally include emollients and penetration enhancers, if desired.

Cream formulations may be prepared as emulsions comprising an oil phaseand aqueous phase, typically including purified water. Components ofcream formulations may include: oil bases, such as petrolatrum, mineraloils, vegetable and animal oils, and triglycerides; cream bases, such aslanolin alcohols, stearic acid, and cetostearyl alcohol; a gel base,such as polyvinyl alcohol; solvents, such as, propylene glycol andpolyethylene glycol; emulsifiers, such as polysorbates, stearates, suchas glyceryl stearate, octylhydroxystearate, polyoxyl stearate, PEGstearyl ethers, isopropyl palmitate, and sorbitan monostearate;stabilizers, such as polysaccharides and sodium sulfite; emollients(i.e. moisturizers), such as medium chain triglycerides, isopropylmyristate, and dimethicone; stiffening agents, such as cetyl alcohol andstearyl alcohol; antimicrobial agents, such as methylparaben,propylparaben, phenoxyethanol, sorbic acid, diazolidinyl urea, andbutylated hydroxyanisole; penetration enhancers, such asN-methylpyrrolidone, propylene glycol, polyethylene glycol monolaurate,and the like; and chelating agents, such as edetate disodium.

Alternatively, the pharmaceutical compositions of the invention areformulated for administration by inhalation. Suitable pharmaceuticalcompositions for administration by inhalation will typically be in theform of an aerosol or a powder. Such compositions are generallyadministered using well-known delivery devices, such as a metered-doseinhaler, a dry powder inhaler, a nebulizer or a similar delivery device.

When administered by inhalation using a pressurized container, thepharmaceutical compositions of the invention will typically comprise theactive ingredient and a suitable propellant, such asdichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas.Additionally, the pharmaceutical composition may be in the form of acapsule or cartridge (made, for example, from gelatin) comprisingcompound 1 and a powder suitable for use in a powder inhaler. Suitablepowder bases include, by way of example, lactose or starch.

The following non-limiting examples illustrate representativepharmaceutical compositions of the present invention.

Tablet Oral Solid Dosage Form

Compound 1, or a pharmaceutically-acceptable salt thereof is dry blendedwith microcrystalline cellulose, polyvinyl pyrrolidone, andcrosscarmellose sodium in a ratio of 4:5:1:1 and compressed into tabletsto provide a unit dosage of, for example, 5 mg, 20 mg or 40 mg activeagent per tablet.

Capsule Oral Solid Dosage Form

Compound 1, or a pharmaceutically-acceptable salt thereof is combinedwith microcrystalline cellulose, polyvinyl pyrrolidone, andcrosscarmellose sodium in a ratio of 4:5:1:1 by wet granulation andloaded into gelatin or hydroxypropyl methylcellulose capsules to providea unit dosage of, for example, 5 mg, 20 mg or 40 mg active agent percapsule.

Liquid Formulation

A liquid formulation comprising compound 1 (0.1%), water (98.9%) andascorbic acid (1.0%) is formed by adding a compound of the invention toa mixture of water and ascorbic acid.

Enteric Coated Oral Dosage Form

Compound 1 is dissolved in an aqueous solution containing polyvinylpyrrolidone and spray coated onto microcrystalline cellulose or sugarbeads in a ratio of 1:5 w/w active agent:beads and then an approximately5% weight gain of an enteric coating comprising an acrylic copolymer isapplied. The enteric coated beads are loaded into gelatin orhydroxypropyl methylcellulose capsules to provide a unit dosage of, forexample, 30 mg active agent per capsule.

Enteric Coated Oral Dosage Form

An enteric coating comprising a combination of Eudragit-L® andEudragit-S®, or hydroxypropyl methylcellulose acetate succinate isapplied to a tablet oral dosage form or a capsule oral dosage formdescribed above.

Aqueous Formulation for Ocular Injection

Each mL of a sterile aqueous suspension includes from 5 mg to 50 mg ofcompound 1, sodium chloride for tonicity, 0.99% (w/v) benzyl alcohol asa preservative, 0.75% carboxymethylcellulose sodium, and 0.04%polysorbate. Sodium hydroxide or hydrochloric acid may be included toadjust pH to 5 to 7.5.

Aqueous Formulation for Ocular Injection

A sterile preservative-free aqueous suspension includes from 5 mg/mL to50 mg/mL of compound 1 in 10 mM sodium phosphate, 40 mM sodium chloride,0.03° % polysorbate 20, and 5% sucrose.

Ointment Formulation for Topical Administration

Compound 1 is combined with petrolatum, C₈-C₁₀ triglyceride,octylhydroxystearate, and N-methylpyrrolidone in a ratio to provide acomposition containing 0.05% to 5% active agent by weight.

Ointment Formulation for Topical Administration

Compound 1 is combined with white petrolatum, propylene glycol, mono-and di-glycerides, paraffin, butylated hydroxytoluene, and edetatecalcium disodium in a ratio to provide a composition containing 0.05% to5% active agent by weight.

Ointment Formulation for Topical Administration

Compound 1 is combined with mineral oil, paraffin, propylene carbonate,white petrolatum and white wax to provide a composition containing 0.05%to 5% active agent by weight.

Cream Formulation for Topical Administration

Mineral oil is combined with compound 1, propylene glycol, isopropylpalmitate, polysorbate 60, cetyl alcohol, sorbitan monostearate,polyoxyl 40 stearate, sorbic acid, methylparaben and propylparaben toform an oil phase, which is combined with purified water by shearblending to provide a composition containing 0.05% to 5% active agent byweight.

Cream Formulation for Topical Administration

A cream formulation comprising compound 1, benzyl alcohol, cetylalcohol, citric acid anhydrous, mono and di-glycerides, oleyl alcohol,propylene glycol, sodium cetostearyl sulphate, sodium hydroxide, stearylalcohol, triglycerides, and water contains 0.05% to 5% active agent byweight.

Cream Formulation for Topical Administration

A cream formulation comprising compound 1, cetostearyl alcohol,isopropyl myristate, propylene glycol, cetomacrogol 1000, dimethicone360, citric acid, sodium citrate, and purified water, with imidurea,methylparaben, and propylparaben, as preservatives, contains 0.05% to 5%active agent by weight.

Dry Powder Composition

Micronized compound 1 (1 g) is blended with milled lactose (25 g). Thisblended mixture is then loaded into individual blisters of a peelableblister pack in an amount sufficient to provide between about 0.1 mg toabout 4 mg of compound 1 per dose. The contents of the blisters areadministered using a dry powder inhaler.

Metered-Dose Inhaler Composition

Micronized compound 1 (10 g) is dispersed in a solution prepared bydissolving lecithin (0.2 g) in demineralized water (200 mL). Theresulting suspension is spray dried and then micronized to form amicronized composition comprising particles having a mean diameter lessthan about 1.5 μm. The micronized composition is then loaded intometered-dose inhaler cartridges containing pressurized1,1,1,2-tetrafluoroethane in an amount sufficient to provide about 0.1mg to about 4 mg of compound 1 per dose when administered by the metereddose inhaler.

Nebulizer Composition

Compound 1 (25 mg) is dissolved in a solution containing 1.5-2.5equivalents of hydrochloric acid, followed by addition of sodiumhydroxide to adjust the pH to 3.5 to 5.5 and 3% by weight of glycerol.The solution is stirred well until all the components are dissolved. Thesolution is administered using a nebulizer device that provides about0.1 mg to about 4 mg of compound 1 per dose.

Utility

Compound 1 has been shown to be a potent inhibitor of the JAK family ofenzymes: JAK1, JAK2, JAK3, and TYK2.

Ocular Diseases

Many ocular diseases have been shown to be associated with elevations ofproinflammatory cytokines that rely on the JAK-STAT pathway. Sincecompound 1 exhibits potent inhibition at all four JAK enzymes, it isexpected to potently inhibit the signaling and pathogenic effects ofnumerous cytokines (such as IL-6, IL-2 and IFN-γ), that signal throughJAK, as well as to prevent the increase in other cytokines (such asMCP-1 and IP-10), whose production is driven by JAK-STAT pathwaysignaling.

In particular, compound 1 exhibited pIC₅₀ values of 6.4 or greater (IC₅₀values of 400 nM or less) for inhibition of IL-6, IL-2, and IFNγsignaling in the cellular assays described in Assays 3 to 6, includingassays registering inhibition of the downstream effects of cytokineelevation.

The pharmacokinetic study of Assay 7 demonstrated sustained exposure inrabbit eyes after a single intravitreal injection and a concentration inplasma at least three orders of magnitude lower than that observed invitreous tissue.

Furthermore, intravitreal dosing of compound 1 has demonstratedsignificant inhibition of IL-6 induced pSTAT3 in the rat retina/choroidtissue as well as significant and sustained inhibition of IFN-γ inducedIP-10 in the rabbit vitreous as well as retina/choroid tissues.Intravitreal dosing of compound 1 has demonstrated significant andsustained inhibition of IFN-γ induced pSTAT1 in the rabbit.

It is expected that sustained ocular JAK inhibition in the absence ofsignificant systemic levels will result in potent, localanti-inflammatory activity in the eye without systemically-drivenadverse effects. Compound 1 is thus expected to be beneficial in anumber of ocular diseases that include, but are not limited to, uveitis,diabetic retinopathy, diabetic macular edema, dry eye disease,age-related macular degeneration, retinal vein occlusion, and atopickeratoconjunctivitis.

In particular, uveitis (Horai and Caspi, J Interferon Cytokine Res,2011, 31, 733-744), diabetic retinopathy (Abcouwer, J Clin Cell Immunol,2013, Suppl 1, 1-12), diabetic macular edema (Sohn et al., AmericanJournal of Opthamology, 2011, 152, 686-694), dry eye disease (Stevensonet al, Arch Ophthalmol, 2012, 130, 90-100), retinal vein occlusion(Shchuko et al, Indian Journal of Ophthalmology, 2015, 63(12), 905-911)and age-related macular degeneration (Knickelbein et al, Int OphthalmolClin, 2015, 55(3), 63-78) are characterized by elevation of certainpro-inflammatory cytokines that signal via the JAK-STAT pathway.Accordingly, compound 1 may be able to alleviate the associated ocularinflammation and reverse disease progression or provide symptom reliefin these diseases.

In one aspect, therefore, the invention provides a method of treating anocular disease in a mammal, the method comprising administering apharmaceutical composition comprising1-(2-(6-(2-ethyl-5-fluoro-4-hydroxyphenyl)-4-fluoro-1H-indazol-3-yl)-1,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridin-5-yl)-2-morpholinoethan-1-oneor a pharmaceutically-acceptable salt thereof and a pharmaceuticalcarrier to the eye of the mammal. In one aspect, the ocular disease isuveitis, diabetic retinopathy, diabetic macular edema, dry eye disease,age-related macular degeneration, retinal vein occlusion or atopickeratoconjunctivitis. In one aspect, the method comprises administeringcompound 1 by intravitreal injection.

Inflammatory Skin Disease

Atopic dermatitis, for example, has been associated with elevation ofproinflammatory cytokines that rely on the JAK-STAT pathway, inparticular, IL-4, IL-5, IL-10, IL-13, and IFNγ. In addition to thecytokine inhibition in cellular assays cited above, compound 1 exhibitedan IC₅₀ value of 13 nM for inhibition of IL-13, as described in Assay 2.Furthermore, model cream and ointment formulations of compound 1 havedemonstrated sustained dermal levels for at least 2 days in mice and atleast 7 days in mini-pig without detectable plasma exposure.

It is expected that sustained dermal levels of compound 1 in the absenceof significant systemic levels will result in potent localanti-inflammatory and anti-pruritic activity in the skin withoutsystemically-driven adverse effects. Therefore, compound 1 is expectedto be beneficial in a number dermal inflammatory or pruritic conditionsthat include, but are not limited to alopecia areata, vitiligo,cutaneous T cell lymphoma, prurigo nodularis, lichen planus, primarylocalized cutaneous amyloidosis, bullous pemphigoid, skin manifestationsof graft versus host disease, pemphigoid, discoid lupus, granulomaannulare, lichen simplex chronicus, vulvar/scrotal/perianal pruritus,lichen sclerosus, post herpetic neuralgia itch, lichen planopilaris, andfoliculitis decalvans. In particular, alopecia areata (Xing et al., NatMed. 2014 September; 20(9): 1043-9), vitiligo (Craiglow et al, JAMADermatol. 2015 October; 151(10): 1110-2), cutaneous T cell lymphoma(Netchiporouk et al., Cell Cycle. 2014; 13(21):3331-5), prurigonodularis (Sonkoly et al., J Allergy Clin Immunol. 2006 February;117(2):411-7), lichen planus (Welz-Kubiak et al., J Immunol Res. 2015;2015:854747), primary localized cutaneous amyloidosis (Tanaka et al., BrJ Dermatol. 2009 December; 161(6):1217-24), bullous pemphigoid(Feliciani et al., Int J Immunopaxthol Pharmacol. 1999 May-August;12(2):55-61), and dermal manifestations of graft versus host disease(Okiyama et al., J Invest Dermatol. 2014 April; 134(4):992-1000) arecharacterized by elevation of certain cytokines that signal via JAKactivation. Accordingly, compound 1 may be able to alleviate associateddermal inflammation or pruritus driven by these cytokines. Inparticular, compound 1 is expected to be useful for the treatment ofatopic dermatitis and other inflammatory skin diseases.

In one aspect, therefore, the invention provides a method of treating aninflammatory skin disease in a mammal (e.g., a human), the methodcomprising applying a pharmaceutical composition comprising1-(2-(6-(2-ethyl-5-fluoro-4-hydroxyphenyl)-4-fluoro-1H-indazol-3-yl)-1,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridin-5-yl)-2-morpholinoethan-1-oneor a pharmaceutically-acceptable salt thereof and a pharmaceuticalcarrier to the skin of the mammal. In one aspect, the inflammatory skindisease is atopic dermatitis.

Compound 1 may also be used in combination with gram positiveantibiotics, such as mupirocin and fusidic acid, to treat inflammatoryskin diseases. In one aspect, therefore, the invention provides a methodof treating an inflammatory skin disease in a mammal, the methodcomprising applying compound 1 and a gram positive antibiotic to theskin of the mammal. In another aspect, the invention provides apharmaceutical composition comprising1-(2-(6-(2-ethyl-5-fluoro-4-hydroxyphenyl)-4-fluoro-1H-indazol-3-yl)-1,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridin-5-yl)-2-morpholinoethan-1-oneor a pharmaceutically-acceptable salt thereof, a gram positiveantibiotic, and a pharmaceutically-acceptable carrier.

Respiratory Diseases

Cytokines which signal through the JAK-STAT pathway, in particular IL-2,IL-3, IL-4, IL-5, IL-6, IL-9, IL-11, IL-13, IL-23, IL-31, IL-27, thymicstromal lymphopoietin (TSLP), interferon-γ (IFNγ) andgranulocyte-macrophage colony-stimulating factor (GM-CSF) have also beenimplicated in asthma inflammation and in other inflammatory respiratorydiseases. As described above, compound 1 has been shown to be a potentinhibitor of the JAK1, JAK2, JAK3, and TYK2 enzymes and has alsodemonstrated potent inhibition of pro-inflammatory cytokines in cellularassays.

The anti-inflammatory activity of JAK inhibitors has been robustlydemonstrated in preclinical models of asthma (Malaviya et al., IntImmunopharmacol, 2010, 10, 829,-836; Matsunaga et al., Biochem andBiophys Res Commun, 2011, 404, 261-267; Kudlacz et al., Eur J Pharmacol,2008, 582, 154-161.) Accordingly, compound 1 is expected to be usefulfor the treatment of inflammatory respiratory disorders, in particular,asthma. Inflammation and fibrosis of the lung is characteristic of otherrespiratory diseases in addition to asthma such as chronic obstructivepulmonary disease (COPD), cystic fibrosis (CF), pneumonitis,interstitial lung diseases (including idiopathic pulmonary fibrosis),acute lung injury, acute respiratory distress syndrome, bronchitis,emphysema, and bronchiolitis obliterans. Compound 1, therefore, is alsoexpected to be useful for the treatment of chronic obstructive pulmonarydisease, cystic fibrosis, pneumonitis, interstitial lung diseases(including idiopathic pulmonary fibrosis), acute lung injury, acuterespiratory distress syndrome, bronchitis, emphysema, bronchiolitisobliterans, and sarcoidosis.

In one aspect, therefore, the invention provides a method of treating arespiratory disease in a mammal (e.g., a human), the method comprisingadministering to the mammal1-(2-(6-(2-ethyl-5-fluoro-4-hydroxyphenyl)-4-fluoro-1H-indazol-3-yl)-1,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridin-5-yl)-2-morpholinoethan-1-oneor a pharmaceutically-acceptable salt thereof.

In one aspect, the respiratory disease is asthma, chronic obstructivepulmonary disease, cystic fibrosis, pneumonitis, chronic obstructivepulmonary disease (COPD), cystic fibrosis (CF), pneumonitis,interstitial lung diseases (including idiopathic pulmonary fibrosis),acute lung injury, acute respiratory distress syndrome, bronchitis,emphysema, bronchiolitis obliterans, or sarcoidosis. In another aspect,the respiratory disease is asthma or chronic obstructive pulmonarydisease.

In a further aspect, the respiratory disease is a lung infection, ahelminthic infection, pulmonary arterial hypertension, sarcoidosis,lymphangioleiomyomatosis, bronchiectasis, or an infiltrative pulmonarydisease. In yet another aspect, the respiratory disease is drug-inducedpneumonitis, fungal induced pneumonitis, allergic bronchopulmonaryaspergillosis, hypersensitivity pneumonitis, eosinophilic granulomatosiswith polyangiitis, idiopathic acute eosinophilic pneumonia, idiopathicchronic eosinophilic pneumonia, hypereosinophilic syndrome, Lofflersyndrome, bronchiolitis obliterans organizing pneumonia, orimmune-checkpoint-inhibitor induced pneumonitis.

The invention further provides a method of treating asthma in a mammal,the method comprising administering to the mammal a pharmaceuticalcomposition comprising1-(2-(6-(2-ethyl-5-fluoro-4-hydroxyphenyl)-4-fluoro-1H-indazol-3-yl)-1,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridin-5-yl)-2-morpholinoethan-1-oneor a pharmaceutically-acceptable salt thereof and apharmaceutically-acceptable carrier.

Compound 1, or a pharmaceutically acceptable salt thereof, may also beuseful to treat eosinophilic lung diseases. Eosinophilic airwayinflammation which is a characteristic feature of diseases collectivelytermed eosinophilic lung diseases (Cottin et al., Clin. Chest. Med.,2016, 37(3), 535-56). Eosinophilic diseases have been associated withIL-4, IL-13 and IL-5 signaling. Eosinophilic lung diseases includeinfections (especially helminthic infections), drug-induced pneumonitis(induced for example by therapeutic drugs such as antibiotics,phenytoin, or 1-tryptophan), fungal-induced pneumonitis (e.g. allergicbronchopulmonary aspergillosis), hypersensitivity pneumonitis andeosinophilic granulomatosis with polyangiitis (formerly known asChurg-Strauss syndrome). Eosinophilic lung diseases of unknown etiologyinclude idiopathic acute eosinophilic pneumonia, idiopathic chroniceosinophilic pneumonia, hypereosinophilic syndrome, and Lofflersyndrome.

Compound 1, or a pharmaceutically acceptable salt thereof, may also beuseful to treat PAH. A polymorphism in the IL-6 gene has been associatedwith elevated IL-6 levels and an increased risk of developing pulmonaryarterial hypertension (PAH) (Fang et al., J Am Soc Hypertens., 2017,11(3), 171-177). Corroborating the role of IL-6 in PAH, inhibition ofthe IL-6 receptor chain gp130 ameliorated the disease in a rat model ofPAH (Huang et al., Can J Cardiol., 2016, 32(11), 1356.e1-1356.e10).

Compound 1, or a pharmaceutically acceptable salt thereof, may also beuseful to treat non-allergic lung diseases such as sarcoidosis, andlymphangioleiomyomatosis. Cytokines such as IFNγ, IL-12 and IL-6 havebeen implicated in a range of non-allergic lung diseases such assarcoidosis, and lymphangioleiomyomatosis (El-Hashemite et al., Am. J.Respir. Cell Mol. Biol., 2005, 33, 227-230, and El-Hashemite et al.,Cancer Res., 2004, 64, 3436-3443).

Compound 1, or a pharmaceutically acceptable salt thereof, may also beuseful to treat bronchiectasis and infiltrative pulmonary diseases whichare diseases associated with chronic neutrophilic inflammation. Certaincytokines are associated with neutrophilic inflammation (e.g. IL-6,IFNγ).

Pathological T cell activation is critical in the etiology of multiplerespiratory diseases. Autoreactive T cells play a role in bronchiolitisobliterans organizing pneumonia (also termed COS). Similar to COS theetiology of lung transplant rejections is linked to an aberrant T cellactivation of the recipients T cells by the transplanted donor lung.Lung transplant rejections may occur early as Primary Graft Dysfunction(PGD), organizing pneumonia (OP), acute rejection (AR) or lymphocyticbronchiolitis (LB) or they may occur years after lung transplantation asChronic Lung Allograft Dysfunction (CLAD). CLAD was previously known asbronchiolitis obliterans (BO) but now is considered a syndrome that canhave different pathological manifestations including BO, restrictiveCLAD (rCLAD or RAS) and neutrophilic allograft dysfunction. Chronic lungallograft dysfunction (CLAD) is a major challenge in long-termmanagement of lung transplant recipients as it causes a transplantedlung to progressively lose functionality (Gauthier et al., CurrTransplant Rep., 2016, 3(3), 185-191). CLAD is poorly responsive totreatment and therefore, there remains a need for effective compoundscapable of preventing or treating this condition. Several JAK-dependentcytokines such as IFNγ and IL-5 are up-regulated in CLAD and lungtransplant rejection (Berastegui et al, Clin Transplant. 2017, 31,e12898). Moreover, high lung levels of CXCR3 chemokines such as CXCL9and CXCL10 which are downstream of JAK-dependent IFN signaling, arelinked to worse outcomes in lung transplant patients (Shino et al, PLOSOne, 2017, 12 (7), e0180281). JAK inhibition has been shown to beeffective in kidney transplant rejection (Vicenti et al., AmericanJournal of Transplantation, 2012, 12, 2446-56). Therefore, compound 1may have the potential to be effective in treating or preventing lungtransplant rejection and CLAD. Similar T cell activation events asdescribed as the basis for lung transplant rejection also are consideredthe main driver of lung graft-versus-host disease (GVHD) which can occurpost hematopoietic stem cell transplants. Similar to CLAD, lung GVHD isa chronic progressive condition with extremely poor outcomes and notreatments are currently approved. A retrospective, multicenter surveystudy of 95 patients with steroid-refractory acute or chronic GVHD whoreceived the systemic JAK inhibitor ruxolitinib as salvage therapydemonstrated complete or partial response to ruxolitinib in the majorityof patients including those with lung GVHD (Zeiser et al, Leukemia,2015, 29, 10, 2062-68). More recently, immune-checkpoint inhibitorinduced pneumonitis, another T cell mediated lung disease emerged withthe increased use of immune-checkpoint inhibitors. In cancer patientstreated with these T cell stimulating agents, fatal pneumonitis candevelop. Compound 1, or a pharmaceutically acceptable salt thereof, hasthe potential to present a novel treatment for these underserved seriousrespiratory diseases.

Gastrointestinal Diseases

As a JAK inhibitor, compound 1, or a pharmaceutically acceptable saltthereof, may also be useful for a variety of other diseases. Compound 1,or a pharmaceutically acceptable salt thereof, may be useful for avariety of gastrointestinal inflammatory indications that include, butare not limited to, inflammatory bowel disease, ulcerative colitis(proctosigmoiditis, pancolitis, ulcerative proctitis and left-sidedcolitis), Crohn's disease, collagenous colitis, lymphocytic colitis,Behcet's disease, celiac disease, immune checkpoint inhibitor inducedcolitis, ileitis, eosinophilic esophagitis, graft versus hostdisease-related colitis, and infectious colitis. Ulcerative colitis(Reimund et al., J Clin Immunology, 1996, 16, 144-150), Crohn's disease(Woywodt et al., Eur J Gastroenterology Hepatology, 1999, 11, 267-276),collagenous colitis (Kumawat et al., Mol Immunology, 2013, 55, 355-364),lymphocytic colitis (Kumawat et al., 2013), eosinophilic esophagitis(Weinbrand-Goichberg et al., Immunol Res, 2013, 56, 249-260), graftversus host disease-related colitis (Coghill et al., Blood, 2001, 117,3268-3276), infectious colitis (Stallmach et al., Int J Colorectal Dis,2004, 19, 308-315), Behcet's disease (Zhou et al., Autoimmun Rev, 2012,11, 699-704), celiac disease (de Nitto et al., World J Gastroenterol,2009, 15, 4609-4614), immune checkpoint inhibitor induced colitis (e.g.,CTLA-4 inhibitor-induced colitis; (Yano et al., J Translation Med, 2014,12, 191), PD-1- or PD-L1-inhibitor-induced colitis), and ileitis(Yamamoto et al., Dig Liver Dis, 2008, 40, 253-259) are characterized byelevation of certain pro-inflammatory cytokine levels. As manypro-inflammatory cytokines signal via JAK activation, compound 1, or apharmaceutically acceptable salt thereof, may be able to alleviate theinflammation and provide symptom relief. In particular, compound 1, or apharmaceutically acceptable salt thereof may be useful for the inductionand maintenance of remission of ulcerative colitis, and for thetreatment of Crohn's disease, immune checkpoint inhibitor inducedcolitis, and the gastrointestinal adverse effects in graft versus hostdisease. In one aspect, therefore, the invention provides a method oftreating a gastrointestinal inflammatory disease in a mammal (e.g., ahuman), the method comprising administering to the mammal, compound 1,or a pharmaceutically acceptable salt thereof, or a pharmaceuticalcomposition comprising a pharmaceutically-acceptable carrier andcompound 1, or a pharmaceutically acceptable salt thereof.

Other Diseases

Compound 1, or a pharmaceutically acceptable salt thereof, may also beuseful to treat other diseases such as other inflammatory diseases,autoimmune diseases or cancers.

Compound 1, or a pharmaceutically acceptable salt thereof, may be usefulto treat one or more of arthritis, rheumatoid arthritis, juvenilerheumatoid arthritis, transplant rejection, xerophthalmia, psoriaticarthritis, diabetes, insulin dependent diabetes, motor neurone disease,myelodysplastic syndrome, pain, sarcopenia, cachexia, septic shock,systemic lupus erythematosus, leukemia, chronic lymphocytic leukemia,chronic myelocytic leukemia, acute lymphoblastic leukemia, acutemyelogenous leukemia, ankylosing spondylitis, myelofibrosis, B-celllymphoma, hepatocellular carcinoma, Hodgkins disease, breast cancer,Multiple myeloma, melanoma, non-Hodgkin lymphoma, non-small-cell lungcancer, ovarian clear cell carcinoma, ovary tumor, pancreas tumor,polycythemia vera, Sjoegrens syndrome, soft tissue sarcoma, sarcoma,splenomegaly, T-cell lymphoma, and thalassemia major.

Combination Therapy

Compounds of the disclosure or a pharmaceutically acceptable saltthereof may be used in combination with one or more agents which act bythe same mechanism or by different mechanisms to treat a disease. Thedifferent agents may be administered sequentially or simultaneously, inseparate compositions or in the same composition. Useful classes ofagents for combination therapy include, but are not limited to,anti-angiogenic, steroid, anti-inflammatory, plasma kallikreininhibitor, placenta growth factor ligand inhibitor, VEGF-A ligandinhibitor, angiopoietin ligand-2 inhibitor, protein tyrosine phosphatasebeta inhibitor, Tek tyrosine kinase receptor stimulator, calcineurininhibitor, VEGF ligand inhibitor, mTOR complex 1 inhibitor, mTORinhibitor, IL-17 antagonist, calmodulin modulator, FGF receptorantagonist, PDGF receptor antagonist, VEGF receptor antagonist, TNFalpha ligand inhibitor, TNF binding agent, proteoglycan 4 stimulator,VEGF-C ligand inhibitor, VEGF-D ligand inhibitor, CD126 antagonist,complement cascade inhibitor, glucocorticoid agonist, complement C5factor inhibitor, cannabinoid receptor antagonist,sphingosine-1-phosphate receptor-1 modulator, sphingosine-1-phosphatereceptor-3 modulator, sphingosine-1-phosphate receptor-4 modulator,sphingosine-1-phosphate receptor-5 modulator, acetaldehyde dehydrogenaseinhibitor, Flt3 tyrosine kinase inhibitor, Kit tyrosine kinaseinhibitor, Protein kinase C inhibitor, adrenocorticotrophic hormoneligand, stromal cell-derived factor 1 ligand inhibitor, immunoglobulinG1 agonist; Interleukin-1 beta ligand inhibitor, mucin stimulator;Nuclear factor kappa B modulator, cytotoxic T-lymphocyte protein-4stimulator, T cell surface glycoprotein CD28 inhibitor, lipoproteinlipase stimulator; PPAR alpha agonist, adenosine A3 receptor agonist,angiotensin II receptor antagonist, VEGF receptor antagonist, interferonbeta ligand, SMAD-2 modulator; TGF beta 1 ligand inhibitor, somatostatinreceptor agonist, IL-2 receptor alpha subunit inhibitor, VEGF-B ligandinhibitor, thymosin beta 4 ligand, angiotensin II AT-1 receptorantagonist, CCR2 chemokine antagonist, membrane copper amine oxidaseinhibitor, CD11a antagonist, ICAM-1 inhibitor, insulin-like growthfactor 1 antagonist, kallikrein inhibitor, fucosyltransferase 6stimulator, GDP fucose synthetase modulator, GHR gene inhibitor, IGF1gene inhibitor, VEGF-1 receptor antagonist, albumin agonist, IL-2antagonist, CSF-1 antagonist; PDGF receptor antagonist, VEGF-2 receptorantagonist, mTOR inhibitor, PPAR alpha agonist, Rho GTPase inhibitor,Rho associated protein kinase inhibitor, complement C3 inhibitor, EGR-1transcription factor inhibitor, nuclear erythroid 2-related factormodulator, nuclear factor kappa B inhibitor, integrin alpha-V/beta-3antagonist, erythropoietin receptor agonist, glucagon-like peptide 1agonist, TNFRSF1A gene stimulator, angiopoietin ligand-2 inhibitor,alpha-2 antiplasmin inhibitor, collagen antagonist, fibronectininhibitor, laminin antagonist, plasmin stimulator, nerve growth factorligand, FGF1 receptor antagonist, FGF3 receptor antagonist, itk tyrosinekinase inhibitor, Lck tyrosine kinase inhibitor, Ltk tyrosine kinasereceptor inhibitor, PDGF receptor alpha antagonist, PDGF receptor betaantagonist, protein tyrosine kinase inhibitor, VEGF-3 receptorantagonist, membrane copper amine oxidase inhibitor, somatostatin 2receptor agonist, somatostatin 4 receptor agonist, somatostatin 5receptor agonist, protein kinase C alpha inhibitor, protein kinase Cbeta inhibitor, protein kinase C delta inhibitor protein kinase Cepsilon inhibitor protein kinase C eta inhibitor, protein kinase C thetainhibitor, ankyrin modulator, mucin stimulator, P2Y2 purinoceptoragonist, gap junction alpha-1 protein inhibitor, CCR3 chemokineantagonist; eotaxin ligand inhibitor, amiloride sensitive sodium channelinhibitor, PDGF receptor antagonist, protein tyrosine kinase inhibitor,retinal pigment epithelium protein inhibitor, matrix metalloproteaseinhibitor, PDGF receptor antagonist, PDGF receptor beta antagonist,PDGF-B ligand inhibitor, growth hormone receptor antagonist, celladhesion molecule inhibitor, integrin modulator, CXCR4 chemokineantagonist, coiled coil domain containing protein inhibitor, Hsp 90modulator, Rho associated protein kinase inhibitor, VEGF gene inhibitor,endoglin inhibitor, CCR3 chemokine antagonist, maxi K potassium channelmodulator, maxi K potassium channel stimulator, PGF2 alpha agonist,prostanoid receptor agonist, voltage gated chloride channel 2 modulator,complement C5a receptor antagonist, inosine monophosphate dehydrogenaseinhibitor, interleukin 18 ligand inhibitor, TRP cation channel M8stimulator, CNTF receptor agonist, TRPV1 gene inhibitor,deoxyribonuclease I stimulator, IRS1 gene inhibitor, Rho associatedprotein kinase inhibitor, poly ADP ribose polymerase 1 inhibitor, polyADP ribose polymerase 2 inhibitor, poly ADP ribose polymerase 3inhibitor, vanilloid VR1 agonist, NFAT5 gene stimulator, Mucinstimulator, Syk tyrosine kinase inhibitor, alpha 2 adrenoceptor agonist,cyclooxygenase inhibitor, amyloid protein deposition inhibitor, glycogensynthase kinase-3 inhibitor, PARP stimulator, tau deposition inhibitor,DDIT4 gene inhibitor, hemoglobin synthesis modulator, interleukin-1 betaligand inhibitor, TNF antagonist, KCNQ voltage-gated potassium channelstimulator, NMDA receptor antagonist, cyclooxygenase 1 inhibitor,cyclooxygenase inhibitor, 5-HT 1a receptor agonist, calcium channelinhibitor, FGF-2 ligand modulator, phosphoinositide 3-kinase inhibitor,CD44 antagonist, hyaluronidase modulator, hyaluronic acid agonist, IL-1antagonist, type I IL-1 receptor antagonist, complement factor Pinhibitor, tubulin antagonist, beta amyloid antagonist, IL2 genestimulator, I-kappa B kinase beta inhibitor, nuclear factor kappa Bmodulator, plasminogen activator inhibitor 1 inhibitor, FGF-2 ligand,protease modulator, and corticotropin modulator.

Specific agents that may be used in combination with the present JAKinhibitor compound include, but are not limited to lanadelumab,aflibercept, RG-7716, AKB-9778, ciclosporin, bevacizumab, everolimus,secukinumab, fluocinolone acetonide, RP-101, squalamine lactate,recombinant human lubricin, OPT-302, sarilumab, dexamethasone,eculizumab, fingolimod, adalimumab, reproxalap, midostaurin,corticotropin, olaptesed pegol, canakinumab, recoflavone, abatacept,fenofibrate, piclidenoson, OpRegen, candesartan, golimumab, pegaptanib,interferon-beta, disitertide, octreotide acetate, anecortave,basiliximab, suprachoroidal triamcinolone acetonide, RGN-259,difluprednate, HL-036, avacincaptad pegol sodium, irbesartan,propagermanium, triamcinolone acetonide, azithromycin, BI-1467335,lifitegrast, loteprednol etabonate, teprotumumab, KVD-001, TZ-101,atesidorsen, Nov-03, bevacizumab, AVA-101, RU-101, voclosporin,vorolanib, sirolimus, choline fenofibrate, VX-210, APL-2, CPC-551,elamipretide. SF-0166, cibinetide, elamipretide, liraglutide, EYS-606,nesvacumab, aflibercept, ocriplasmin, filgotinib, cenegermin, adipocell,brolucizumab, ranibizumab, aflibercept, padeliporfin photodynamictherapy, pazopanib, ASP-8232, veldoreotide, sotrastaurin, abiciparpegol, diquafosol tetrasodium, HCB-1019, conbercept, bertilimumab,SHP-659, THR-317, ALK-001, PAN-90806, interferon alfa-2b, fluocinolone,sunitinib malate, emixustat, hI-con1, TB-403, minocycline, MA09-hRPEcells, pegpleranib sodium, pegvisomant, luminate, burixafor, H-1129,carotuximab, AXP-1275, ranibizumab, isopropyl unoprostone, tesidolumab,enteric-coated mycophenolate sodium, tadekinig alfa, triamcinoloneacetonide, cyclosporine, ST-266, AVX-012, NT-501-ECT, tivanisiran,verteporfin, dornase alfa, aganirsen, ripasudil, rucaparib phosphate,zucapsaicin, tetrathiomolybdate, diclofenac, LHA-510, AGN-195263,tacrolimus, rebamipide, R-348, brimonidine tartrate, vizomitin, T-89,LME-636, BI-1026706, rimexolone, tobramycin, TOP-1630, talaporfin,bromfenac sodium, triamcinolone acetonide, davunetide, loteprednoletabonate, XED-60, EG-Mirotin, APD-209, adenovir, PF-04523655,hydroxycarbamide, navamepent, retinalamin, CNTO-2476, ranibizumab,flupirtine, B27PD, S-646240, GLY-230, hydralazine, nepafenac, DexNP,Trehalose, hyaluronic acid, dexamethasone-Ca sustained-release depot,naluzotan, hyaluronidase, sodium hyaluronate, isunakinra, somatostatin,CLG-561, OC-10X, UCA-002, recombinant human epidermal growth factor,pemirolast, VM-100, MB-11316, monosodium alpha luminol, ranibizumab,IMD-1041, LMG-324, HE-10, cinhyaluronate sodium, BDM-E, mesenchymalprecursor cells, disulfiram, CTC-96, PG-101, Beifushu, chymotrypsin.

Also provided, herein, is a pharmaceutical composition comprisingcompound 1, or a pharmaceutically acceptable salt thereof, and one ormore other therapeutic agents. The therapeutic agent may be selectedfrom the class of agents specified above and from the list of specificagent described above. In some embodiments, the pharmaceuticalcomposition is suitable for ocular delivery. In some embodiments, thepharmaceutical composition is a liquid or suspension composition.

Further, in a method aspect, the invention provides a method of treatinga disease or disorder in a mammal comprising administering to the mammalcompound 1, or a pharmaceutically acceptable salt thereof, and one ormore other therapeutic agents.

When used in combination therapy, the agents may be formulated in asingle pharmaceutical composition, or the agents may be provided inseparate compositions that are administered simultaneously or atseparate times, by the same or by different routes of administration.Such compositions can be packaged separately or may be packaged togetheras a kit. The two or more therapeutic agents in the kit may beadministered by the same route of administration or by different routesof administration.

EXAMPLES

The following synthetic and biological examples are offered toillustrate the invention, and are not to be construed in any way aslimiting the scope of the invention.

In the examples below, the following abbreviations have the followingmeanings unless otherwise indicated. Abbreviations not defined belowhave their generally accepted meanings.

-   -   ACN=acetonitrile    -   DCC=dicyclohexylcarbodiimide    -   DIPEA=N,N-diisopropylethylamine    -   DMAc=dimethylacetamide    -   DMF=N,N-dimethylformamide    -   DMSO=dimethyl sulfoxide    -   EtOAc=ethyl acetate    -   HATU=N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)uronium        hexafluorophosphate    -   LDA=lithium diisopropylamide    -   min=minute(s)    -   MTBE=methyl tert-butyl ether    -   NBS=N-bromosuccinimide    -   NMP=N-Methyl-2-pyrrolidone    -   RT=room temperature    -   THF=tetrahydrofuran    -   bis(pinacolato)diboron=4,4,5,5,4′,4′,5′,5′-octamethyl-[2,2′]bi[[1,3,2]dioxaborolanyl]    -   Pd(dppf)Cl₂—CH₂Cl₂=dichloro(1,1′-bis(diphenylphosphino)-ferrocene)-dipalladium(II)        complex with dichloromethane

Reagents and solvents were purchased from commercial suppliers (Aldrich,Fluka, Sigma, etc.), and used without further purification. Progress ofreaction mixtures was monitored by thin layer chromatography (TLC),analytical high performance liquid chromatography (anal. HPLC), and massspectrometry. Reaction mixtures were worked up as described specificallyin each reaction; commonly they were purified by extraction and otherpurification methods such as temperature-, and solvent-dependentcrystallization, and precipitation. In addition, reaction mixtures wereroutinely purified by column chromatography or by preparative HPLC,typically using C18 or BDS column packings and conventional eluents.Typical preparative HPLC conditions are described below.

Characterization of reaction products was routinely carried out by massand ¹H-NMR spectrometry. For NMR analysis, samples were dissolved indeuterated solvent (such as CD₃OD, CDCl₃, or d₆-DMSO), and ¹H-NMRspectra were acquired with a Varian Gemini 2000 instrument (400 MHz)under standard observation conditions. Mass spectrometric identificationof compounds was performed by an electrospray ionization method (ESMS)with an Applied Biosystems (Foster City, Calif.) model API 150 EXinstrument or a Waters (Milford, Mass.) 3100 instrument, coupled toautopurification systems.

Preparative HPLC Conditions Column: C18, 5 μm. 21.2×150 mm or C18, 5 μm21×250 or

-   -   C14, 5 μm 21×150 mm        Column temperature: Room Temperature        Flow rate: 20.0 mL/min

Mobile Phases: A=Water+0.05% TFA

-   -   B=ACN+0.05% TFA,        Injection volume: (100-1500 μL)        Detector wavelength: 214 nm

Crude compounds were dissolved in 1:1 water:acetic acid at about 50mg/mL. A 4 minute analytical scale test run was carried out using a2.1×50 mm C18 column followed by a 15 or 20 minute preparative scale runusing 100 μL injection with the gradient based on the % B retention ofthe analytical scale test run. Exact gradients were sample dependent.Samples with close running impurities were checked with a 21×250 mm C18column and/or a 21×150 mm C14 column for best separation. Fractionscontaining desired product were identified by mass spectrometricanalysis.

Preparation 1:2-(4-(benzyloxy)-2-ethyl-5-fluorophenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane(1-5)

(a) 2-(Benzyloxy)-4-bromo-1-fluorobenzene (1-2)

Two reactions were carried out in parallel and combined for work-up. Amixture of 5-bromo-2-fluorophenol (1-1) (850 g, 4.5 mol), benzyl bromide(837 g, 4.9 mol) and potassium carbonate (923 g, 6.7 mol) in ACN (5 L)was stirred at 20° C. for 12 h. The reactions were combined andconcentrated, diluted with water (8 L), and extracted with EtOAc (3×3L). The organic layer was separated, washed with brine (3 L), dried oversodium sulfate and concentrated. The crude product was purified througha silica gel pad (eluted with 3:1 petroleum ether:EtOAc) to give thetitle intermediate (1.83 kg, 73% yield) as a white solid. ¹H NMR (400MHz, CDCl₃) δ 7.38-7.46 (m, 5H), 7.15 (dd, J=7.6, 2.0 Hz, 1H), 6.98-7.15(m, 1H), 5.12 (s, 2H).

(b) 2-(Benzyloxy)-4-ethyl-1-fluorobenzene (1-3)

Six reactions were carried out in parallel and combined for work-up. Toa solution of the product of the previous step (200 g, 711 mmol) in THF(100 mL) was added potassium carbonate (197 g, 1.4 mol). The reactionmixture was purged with nitrogen 3 times, followed by addition ofPd(dppf)Cl₂—CH₂Cl₂ (11.6 g, 14.2 mmol). The reaction mixture was cooledto 0° C., diethylzinc (1 M, 1.07 L) was added drop-wise, and thereaction mixture was stirred at 70° C. for 1 h. The reactions werecombined, cooled to 20° C. and poured into water (7 L) slowly. To themixture was added aq. 4 M HCl to pH 6. The organic layer was separated,and the aqueous phase was extracted with EtOAc (3×2 L). The combinedorganic layer was washed with brine (5 L), dried over sodium sulfate,concentrated, and purified through a silica gel pad (eluted with 50:1petroleum ether:EtOAc)) to give the title intermediate (900 g, 92%yield) as a light yellow oil. ¹H NMR (400 MHz, CDCl₃) δ 7.29-7.43 (m,5H), 6.94-6.97 (m, 1H), 6.82 (d, J=8.0 Hz, 1H), 6.70 (m, 1H), 5.09 (s,2H), 2.52-2.58 (m, 2H), 1.17 (t, J=7.6 Hz, 3H).

(c) 1-(Benzyloxy)-4-bromo-5-ethyl-2-fluorobenzene (1-4)

Four reactions were carried out in parallel and combined. To a solutionof 2-(benzyloxy)-4-ethyl-1-fluorobenzene (1-3) (293 g, 1.3 mol) in ACN(1 L) was added NBS (249 g, 1.4 mol) in portions at 20° C. The reactionmixture was stirred at 20° C. for 2 h. The reaction mixtures werecombined and concentrated. The residue was diluted with water (5 L) andextracted with EtOAc (2×5 L). The organic phase was washed with brine (4L), dried over anhydrous sodium sulfate, filtered and concentrated invacuo. The crude product was purified by silica gel chromatography(eluted with petroleum ether:EtOAc 100:1-10:1) to give the titleintermediate (1.4 kg, 89% yield) as light yellow oil. ¹H NMR (400 MHz,CDCl₃) δ 7.29-7.38 (m, 5H), 7.2 (d, J=10.4 Hz, 1H), 6.8 (d, J=8.8 Hz,1H), 5.06 (s, 2H), 2.6 (q, J=7.6 Hz, 2H), 1.1 (t, J=7.6 Hz, 3H).

(d)2-(4-(benzyloxy)-2-ethyl-5-fluorophenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane(1-5)

Seven reactions were carried out in parallel and combined for work-up.To a solution of the product of the previous step (200 g, 647 mmol) indioxane (2 L) was added potassium acetate (190 g, 1.9 mol),bis(pinacolato)diboron (181 g, 712 mmol), and Pd(dppf)Cl₂—CH₂Cl₂ (10.6g, 12.9 mmol) under nitrogen at 20° C. The mixture was stirred at 120°C. for 2 h. The reaction mixtures were combined, concentrated, dilutedwith water (5 L), and extracted with EtOAc (3×4 L). The combined organicphase was dried with anhydrous sodium sulfate, filtered and concentratedin vacuo. The crude product was purified by silica gel chromatography(eluted with petroleum ether:EtOAc 1:0-5:1) to give the title compound(1.35 kg, 84% yield) as a white solid. ¹H NMR (400 MHz, CDCl₃) δ7.33-7.51 (m, 6H), 6.82 (d, J=7.6 Hz, 1H), 5.17 (s, 2H), 2.85 (q,0.1=7.6 Hz, 2H), 1.33 (s, 12H), 1.15 (t, J=7.6 Hz, 3H).

Preparation 2: 1-Benzyl-4-imino-1,4-dihydropyridin-3-amine (2)

To a solution of pyridine-3,4-diamine (400 g, 3.67 mol) in ACN (3 L) wasadded benzyl bromide (596 g, 3.49 mol) in portions at 0° C. and thereaction mixture was stirred for 30 min and then at 20° C. for 12 h, andfiltered. The filter cake was washed with ACN (500 mL) and dried to givethe HBr salt of the title compound (600 g, 2.14 mol, 58% yield) as awhite powder. ¹H NMR (400 MHz, MeOD) δ 7.83 (d, J=5.6 Hz, 1H), 7.64 (s,1H), 7.32-7.40 (m, 5H), 6.76 (d, J=6.8 Hz, 1H), 5.28 (s, 2H).

Preparation 3:6-(4-(Benzyloxy)-2-ethyl-5-fluorophenyl)-4-fluoro-1H-indazole-3-carbaldehyde(3)

(a) 1-(4-Bromo-2,6-difluorophenyl)-2,2-diethoxyethan-1-one (3-1)

Nine reactions were carried out in parallel and combined for work-up. Asolution of 1-bromo-3,5-difluorobenzene (100 g, 518 mmol) in THF (700mL) was degassed and purged with nitrogen three times. Then 2 M LDA (311mL) was added at −70° C. and the reaction mixture was stirred at −70° C.for 0.5 h under nitrogen. A solution of ethyl 2,2-diethoxyacetate (96 g,544 mmol) in THF (200 mL) was added drop-wise at −70° C. under nitrogenand the reaction mixture was stirred for 1 h. The reactions werecombined and poured into ice saturated ammonium chloride (10 L) inportions and extracted with EtOAc (3×3 L). The organic layer wasseparated, washed with brine (5 L), dried over sodium sulfate,concentrated, and purified by silica gel chromatography (eluted withpetroleum ether EtOAc 1:0-100:1) to give the title compound (1.26 kg,84% yield) as a yellow oil. ¹H NMR (400 MHz, CDCl₃) δ 7.12 (d, J=7.2 Hz,2H), 5.15 (s, 1H), 3.61-3.7 (m, 4H), 1.2 (t, J=7.2 Hz, 6H).

(b)1-(4′-(benzyloxy)-2′-ethyl-3,5,5′-trifluoro-[1,1′-biphenyl]-4-yl)-2,2-diethoxyethan-1-one(3-2)

Five reactions were carried out in parallel and combined for work-up. Toa mixture of 1-(4-bromo-2,6-difluorophenyl)-2,2-diethoxyethan-1-one(3-1) (189 g, 586 mmol) in ethanol (150 mL) and toluene (1.5 L) wasadded water (150 mL), sodium carbonate (84.8 g, 800 mmol), and2-(4-(benzyloxy)-2-ethyl-5-fluorophenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane(1-5) (190 g, 533 mmol) at 20° C. The suspension was degassed undervacuum and purged with nitrogen several times. Pd(dppf)Cl₂—CH₂Cl₂ (13 g,16 mmol) was added and the reaction mixture was purged with nitrogenseveral times and stirred at 120° C. for 2 h. The reactions werecombined, cooled to 20° C., poured into water (5 L) and extracted withEtOAc (3×4 L). The combined organic layers were washed with brine (5 L),dried over sodium sulfate, filtered, concentrated, and purified bysilica gel chromatography (eluted with petroleum ether:EtOAc 100:1-5:1)to give the title intermediate (880 g, 70% yield) as a yellow oil. ¹HNMR (400 MHz, CDCl₃) δ 7.36-7.48 (m, 5H), 6.94-6.96 (m, 2H), 6.86-6.92(m, 2H), 5.29 (s, 1H), 5.19 (s, 2H), 3.67-3.77 (m, 4H), 2.52 (q, J=7.6Hz, 2H), 1.25 (t, J=6.8 Hz, 6H), 1.07 (t, J=7.2 Hz, 3H).

(c)6-(4-(benzyloxy)-2-ethyl-5-fluorophenyl)-3-(diethoxymethyl)-4-fluoro-1H-indazole(3-3)

Four reactions were carried out in parallel and combined for work-up. Toa solution of the product of the previous step (220 g, 466 mmol) in THF(2 L) was added hydrazine monohydrate (47.6 g, 931 mmol) at 20° C. Thereaction mixture was stirred at 100° C. for 12 h. Four reactions werecombined and cooled to 20° C. and concentrated. The residue wasdissolved in EtOAc (5 L) and washed with 0.1 M HCl (2×1.5 L). Thecombined organic layers were washed with brine (1.5 L), dried oversodium sulfate, filtered and concentrated to give the title intermediate(900 g, crude) as yellow gum, which was used directly in the next step.¹H NMR (400 MHz, CDCl₃) δ 7.36-7.48 (m, 5H), 6.94-6.96 (m, 2H),6.86-6.92 (m, 2H), 5.29 (s, 1H), 5.19 (s, 2H), 3.67-3.77 (m, 4H), 2.52(q, J=7.6 Hz, 2H), 1.25 (t, J=6.8 Hz, 6H), 1.07 (t, J=7.2 Hz, 3H).

(d)6-(4-(Benzyloxy)-2-ethyl-5-fluorophenyl)-4-fluoro-1H-indazole-3-carbaldehyde(3)

Three reactions were carried out in parallel and combined for work-up.To a solution of the product of the previous step (300 g, 643 mmol) inacetone (1.5 L) was added 4 M HCl (16 mL) drop-wise at 20° C. and thereaction mixture was stirred at 20° C. for 0.17 h. The reactions werecombined, concentrated, diluted with MTBE (1 L), and filtered. Thefilter cake was washed with MTBE (2×300 mL) and dried under reducedpressure to give the title intermediate (705 g, crude) as a yellowsolid, which was used directly in the next step. (m/z): [M+H]⁺ calcd forC₂₃H₁₈F₂N₂O₂ 393.13 found 393.1. ¹H NMR (400 MHz, DMSO-d₆) δ 14.51 (s,1H), 10.17 (d, J=3.6 Hz, 1H), 7.50 (d, J=7.2 Hz, 2H), 7.40-7.42 (m, 4H),7.24 (d, J=8.4 Hz, 1H), 7.15 (d, J=12.4 Hz, 1H), 7.06 (d, J=8.4 Hz, 1H),5.25 (s, 2H), 2.52-2.53 (m, 2H), 1.03 (t, J=7.6 Hz, 3H).

Preparation 4:5-Benzyl-2-(6-(4-(benzyloxy)-2-ethyl-5-fluorophenyl)-4-fluoro-1H-indazol-3-yl)-5H-imidazo[4,5-c]pyridine(4)

Four reactions were carried out in parallel and combined for work-up. Toa solution of6-(4-(benzyloxy)-2-ethyl-5-fluorophenyl)-4-fluoro-1H-indazole-3-carbaldehyde(3), the product of Preparation 3 (172 g, 440 mmol) in DMF (1.1 L) wasadded sodium bisulfite (68.6 g, 659 mmol) and1-benzyl-4-imino-1,4-dihydropyridin-3-amine (2) (136 g, 484 mmol) at 20°C. and the reaction mixture was stirred at 150° C. for 2 h. Fourreactions were combined and the reaction mixture was concentrated underreduced pressure. The residue was poured into water (10 L) and filtered.The filter cake was dried under reduced pressure to give the titleintermediate (990 g, crude) as a yellow solid, which was used directlywithout purification. (m z): [M+H]⁺ calcd for C₃₅H₂₇F₂N₅O 572.2 found572.3.

Preparation 5:5-Benzyl-2-(6-(4-(benzyloxy)-2-ethyl-5-fluorophenyl)-4-fluoro-1H-indazol-3-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine(5)

Three reactions were carried out in parallel and combined for work-up.To a mixture of5-benzyl-2-(6-(4-(benzyloxy)-2-ethyl-5-fluorophenyl)-4-fluoro-1H-indazol-3-yl)-5H-imidazo[4,5-c]pyridine(4), the product of Preparation 4 (330 g, 577 mmol) in methanol (1.5 L)and THF (1 L) was added sodium borohydride (267 g, 6.9 mol) in portionsat 20° C. and the reaction mixture was stirred at 20° C. for 24 h. Threereactions were combined and the reaction mixture was added to water (10L), stirred for 10 min, and filtered. The filtrate was extracted withEtOAc (2×5 L) and the combined organic phase was dried with anhydroussodium sulfate, filtered and concentrated under vacuum. The crudeproduct was diluted with EtOAc (2 L), stirred for 30 min, and filtered.The filter cake was washed with MTBE (3×200 mL) to give the titleintermediate (275 g, 28% yield) as a light yellow solid. (m/z): [M+H]⁺calcd for C₃₅H₃₁F₂N₅O 576.25 found 576.3. ¹H NMR (400 MHz, DMSO-d₆) δ7.50-7.52 (m, 2H), 7.35-7.43 (m, 7H), 7.23-7.25 (m, 3H), 7.15 (d, J=12.0Hz, 1H), 6.81 (d, J=12.0 Hz, 1H), 5.25 (s, 2H), 3.72 (s, 2H), 3.43 (br.s, 2H), 2.78 (br. s, 2H), 2.66 (br. s, 2H), 2.55 (q, 2H), 1.04 (t, J=7.6Hz, 3H).

Preparation 6:5-Ethyl-2-fluoro-4-(4-fluoro-3-(4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)phenol(6)

Five reactions were carried out in parallel and combined for work-up. Toa mixture of5-benzyl-2-(6-(4-(benzyloxy)-2-ethyl-5-fluorophenyl)-4-fluoro-1H-indazol-3-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine(5), the product of Preparation 5 (55 g, 95.5 mmol) in THF (500 mL) andmethanol (500 mL) was added palladium on carbon (15 g, 9.6 mmol) and aq.12 M HCl (10 mL). The suspension was degassed under vacuum, purged withhydrogen several times and stirred under hydrogen (50 psi) at 50° C. for12 h. The reactions were combined and the reaction mixture was filtered.The filtrate was concentrated under vacuum to provide the HCl salt ofthe title intermediate (150 g crude) as an off-white solid. (m/z):[M+H]⁺ calcd for C₂₁H₁₉F₂N₅O 396.16 found 396.2. ¹H NMR (400 MHz, MeOD)δ 7.43 (s, 1H), 7.07 (d, J=11.6 Hz, 1H), 6.97 (d, J=11.6 Hz, 1H), 6.91(d, J=8.8 Hz, 1H), 4.57 (s, 2H), 3.74 (s, 2H), 3.24 (s, 2H), 2.55 (q,J=7.6 Hz, 2H), 1.08 (t, J=7.6 Hz, 3H).

Preparation 7: 2,5-dioxopyrrolidin-1-yl 2-morpholinoacetate (7′)

(a) tert-Butyl 2-morpholinoacetate (7-1)

To a mixture of morpholine (160 g, 1.84 mol) and potassium carbonate(381 g, 2.76 mol) in THF (3 L) was added tert-butyl 2-bromoacetate (341g, 1.75 mol) slowly at 0° C. The reaction mixture was stirred for 30 minand then at 20° C. for 12 h, and concentrated. Water (1.5 L) was addedand the reaction mixture was extracted with EtOAc (3×1 L). The organiclayer was separated, washed with brine (500 mL), dried over sodiumsulfate and concentrated to give the title intermediate (300 g, 81%yield) as a yellow oil. ¹H NMR (400 MHz, CDCl₃) δ 3.74 (t, J=4.8 Hz,4H), 3.10 (s, 2H), 2.57 (t, J=4.8 Hz, 4H), 1.46 (s, 9H).

(b) 2-Morpholinoacetic acid (7″)

A mixture of the product of the previous step (7-1) (300 g, 1.49 mol) in3 M HCl in dioxane (2.0 L) was stirred at 20° C. for 12 h andconcentrated to give the HCl salt of the title compound (270 g, 99%yield) as a pale solid, which was used directly in the next step ¹H NMR(400 MHz, MeOD) δ 4.13 (s, 2H), 3.93 (br. s, 4H), 3.64 (br. s, 4H).

(c) 2,5-dioxopyrrolidin-1-yl 2-morpholinoacetate (7′)

A mixture of the product of the previous step (150 g, 826 mmol),1-hydroxypyrrolidine-2,5-dione (95 g, 826 mmol), DCC (256 g, 1.24 mol)and DIPEA (160 g, 1.24 mol) in DCM (2 L) was stirred at 15° C. for 12 hand filtered. The filtrate was concentrated and washed with EtOAc (800mL). The solid was collected by filtration and concentrated to give thetitle compound (150 g, 75% yield) as a white solid. ¹H NMR (400 MHz,DMSO-d₆) δ 3.68 (s, 2H), 3.58 (t, J=4.8 Hz, 4H), 2.82 (s, 4H), 2.57 (t,J=5.2 Hz, 4H).

Example 1:1-(2-(6-(2-Ethyl-5-fluoro-4-hydroxyphenyl)-4-fluoro-1H-indazol-3-yl)-1,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridin-5-yl)-2-morpholinoethan-1-one(1)

A mixture of5-ethyl-2-fluoro-4-(4-fluoro-3-(4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)phenol(6) 2 HCl (100 g, 214 mmol), 2,5-dioxopyrrolidin-1-yl2-morpholinoacetate (7′) (67.2 g, 278 mmol), and DIPEA (69 g, 534 mmol)in DMF (600 mL) was stirred at 15° C. for 12 h and filtered. Thesolution was purified by reverse-phase chromatography (Agela FLEXATMFS-1L instrument; 2 kg Agela C18 DAC column; 200 g sample dissolved inDMF (900 mL); flow rate 300 mL/min; solvent A water, solvent B ACN;gradient (% B, time (min): 0/15, 0-40/45, 40/50) to afford the titlecompound (50.0 g, 44.8% yield) as a light yellow solid. (m/z): [M+H]⁺calcd for C₂₇H₂₈F₂N₆O₃ 523.22 found 523.0. ¹H NMR (400 MHz, MeOD) δ 7.22(s, 1H), 6.80-6.96 (m, 3H), 4.68-4.78 (m, 2H), 3.96 (s, 2H), 3.65-3.95(m, 4H), 3.35-3.38 (m, 2H), 2.77-2.92 (m, 2H), 2.52-2.56 (m, 6H), 1.06(t, J=7.6 Hz, 3H).

Example 2: Crystalline1-(2-(6-(2-Ethyl-5-fluoro-4-hydroxyphenyl)-4-fluoro-1H-indazol-3-yl)-1,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridin-5-yl)-2-morpholinoethan-1-one(1) Form 1

To a 250 mL flask was added1-(2-(6-(2-ethyl-5-fluoro-4-hydroxyphenyl)-4-fluoro-1H-indazol-3-yl)-1,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridin-5-yl)-2-morpholinoethan-1-one(1), the product of Example 1 (5 g) and ethanol (50 mL) and the reactionmixture was stirred at 50-80° C. for 10 min and then ACN (75 mL) wasadded slowly at 50-80° C. followed by seeds from Example 3. The reactionmixture was stirred at 20-25° C. for 18 h. The resulting solid wascollected by filtration and dried at 50° C. under vacuum for 18 h toprovide the title compound Form 1 (3.6 g, 72% yield)

Example 3: Crystalline1-(2-(6-(2-Ethyl-5-fluoro-4-hydroxyphenyl)-4-fluoro-1H-indazol-3-yl)-1,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridin-5-yl)-2-morpholinoethan-1-one(1) Form 1

Compound 1, the product of Example 1, (1 g) was added to ethanol (10 mL)and heated to dissolution. Acetonitrile (10 mL) was added and thereaction mixture was stirred and warmed and then stirred at RT for 16 h,filtered, and dried at 50° C. under vacuum for 18 h to provide the titlecompound Form 1 (0.23 g).

Example 4:1-(2-(6-(2-Ethyl-5-fluoro-4-hydroxyphenyl)-4-fluoro-1H-indazol-3-yl)-1,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridin-5-yl)-2-morpholinoethan-1-one(1)

N,N-diisopropylethylamine (0.298 mL, 1.707 mmol) was added to a solutionof5-ethyl-2-fluoro-4-(4-fluoro-3-(4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)phenol(135 mg, 0.341 mmol) (6), HATU (156 mg, 0.410 mmol) and2-morpholinoacetic acid (7″) (54.5 mg, 0.376 mmol) in DMF (0.5 mL) andthe reaction mixture was stirred at RT for 24 h. Lithium hydroxide (49.1mg, 2.049 mmol) was added and the reaction mixture was stirred at 65° C.for 1 h, and concentrated in vacuo to yield a clear yellow liquid. Thecrude liquid was purified via preparatory HPLC to yield the TFA salt ofthe title compound (142 mg, 0.223 mmol, 65.3% yield) as a beige solid.

Example 5: Crystalline1-(2-(6-(2-Ethyl-5-fluoro-4-hydroxyphenyl)-4-fluoro-1H-indazol-3-yl)-1,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridin-5-yl)-2-morpholinoethan-1-one(1) Form 2

The compound 1 of example 1 (2.5 g) was dissolved in DMSO (5 mL) at 60°C. Once a homogenous solution was obtained, MeOH (2.5 mL) was added tothe solution. The homogenous mixture was added dropwise over 30 min to apremixed solution of MeOH (12.75 mL) and H₂O (11.25 mL) at 75° C. Oncethe mixture had been fully added, the combined mixture was allowed tostir at 75° C. for 1 h while a crystalline slurry formed. H₂O (36 mL)was added dropwise over 2 h at 75° C. After the H₂O charge was complete,the slurry was stirred at 75° C. for 1 h, then slowly cooled to 20° C.over 6 h. The slurry was held at 20° C. for an additional 10 h beforebeing filtered, washed with 70% H₂O/MeOH (10 mL), dried at 50° C. undervacuum for 18 h to provide the title compound Form 2 (2.13 g).

Properties of the Solid Forms of the Invention

Samples of the two anhydrous forms, Form 1 and Form 2 of1-(2-(6-(2-Ethyl-5-fluoro-4-hydroxyphenyl)-4-fluoro-1H-indazol-3-yl)-1,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridin-5-yl)-2-morpholinoethan-1-one (1) of Examples 2 and 5,respectively, were analyzed by powder X-ray diffraction (PXRD),differential scanning calorimetry (DSC), thermogravimetric analysis(TGA), dynamic moisture sorption (DMS), and polarized light microscopyimage. Form 2 was also analyzed by single crystal x-ray diffraction.

Example 6: Powder X-Ray Diffraction

The powder X-ray diffraction patterns of FIGS. 1 and 6 were obtainedwith a Bruker D8-Advance X-ray diffractometer using Cu-Kα radiation(λ=1.54051 Å) with output voltage of 45 kV and current of 40 mA. Theinstrument was operated in Bragg-Brentano geometry with incident,divergence, and scattering slits set to maximize the intensity at thesample. For measurement, a small amount of powder (5-25 mg) was gentlypressed onto a sample holder to form a smooth surface and subjected toX-ray exposure. The samples were scanned in 2θ-2θ mode from 2° to 35° in2θ with a step size of 0.020 and a scan speed of 0.30° seconds per step.The data acquisition was controlled by Bruker DiffracSuite measurementsoftware and analyzed by Jade software (version 7.5.1). The instrumentwas calibrated with a corundum standard, within ±0.02° two-theta angle.Observed PXRD two-theta peak positions and d-spacings are shown inTables 1 and 2, respectively for crystalline Form 1 and the crystallineForm 2.

TABLE 1 PXRD Data for Crystalline Form 1 2-Theta d (Å) Area A % 7.6911.49 5570 7.30 8.16 10.83 36847 48.60 8.97 9.85 75877 100.00 10.66 8.297323 9.70 11.46 7.72 5841 7.70 11.91 7.43 1496 2.00 15.29 5.79 7115 9.4015.80 5.60 7841 10.30 16.70 5.31 14679 19.30 17.02 5.20 8024 10.60 18.004.92 17834 23.50 18.83 4.71 2658 3.50 20.18 4.40 18636 24.60 22.39 3.977067 9.30 22.98 3.87 9029 11.90 24.89 3.57 8561 11.30 26.54 3.36 783110.30

TABLE 2 PXRD Data for the Crystalline Form 2 2-Theta d (Å) Area A %10.61 8.33 706299 100.00 10.85 8.15 192921 27.30 11.84 7.47 487816 69.1013.32 6.64 97980 13.90 14.94 5.93 519386 73.50 16.14 5.49 110314 15.6016.35 5.42 75483 10.70 17.69 5.01 197341 27.90 18.26 4.85 445270 63.0018.43 4.81 152845 21.60 19.06 4.65 564088 79.90 19.20 4.62 427174 60.5019.49 4.55 266328 37.70 20.72 4.28 72244 10.20 21.10 4.21 236517 33.5021.94 4.05 287485 40.70 22.64 3.93 121406 17.20 23.64 3.76 152841 21.6025.19 3.53 68220 9.70 28.08 3.17 139597 19.80

Example 7: Thermal Analysis

Differential scanning calorimetry (DSC) was performed using a TAInstruments Model Q-100 module with a Thermal Analyst controller. Datawere collected and analyzed using TA Instruments Thermal Analysissoftware. A sample of each crystalline form was accurately weighed intoa covered aluminum pan. After a 5 minute isothermal equilibration periodat 5° C., the sample was heated using a linear heating ramp of 10°C./min from 0° C. to 300° C.

A representative DSC thermogram of the Form 1 crystalline free form ofthe invention is shown in FIG. 7. The differential scanning calorimetry(DSC) trace recorded at a heating rate of 10° C. per minute exhibits apeak in endothermic heat flow, identified as a melt transition, in therange of about 210° C. to about 234° C., or in the range of betweenabout 215° C. to about 229° C., or in the range of between about 220° C.to about 224° C. The crystalline form is characterized by a differentialscanning calorimetry trace recorded at a heating rate of 10° C. perminute which shows a maximum in endothermic heat flow with a peak atabout 221.7° C., or at 221.7±3° C.

A representative DSC thermogram of the Form 2 crystalline free form ofthe invention is shown in FIG. 2. The differential scanning calorimetry(DSC) trace recorded at a heating rate of 10° C. per minute exhibits apeak in endothermic heat flow, identified as a melt transition, in therange of about 268° C. to about 277° C., or in the range of betweenabout 270° C. to about 275° C., or in the range of between about 271° C.to about 274° C. The crystalline form is characterized by a differentialscanning calorimetry trace recorded at a heating rate of 10° C. perminute which shows a maximum in endothermic heat flow with a peak atabout 272.6° C., or at 272.6±2° C.

Thermogravimetric analysis (TGA) measurements were performed using a TAInstruments Model Q-50 module equipped with high resolution capability.Data were collected using TA Instruments Thermal Analyst controller andanalyzed using TA Instruments Universal Analysis software. A weighedsample was placed onto a platinum pan and scanned with a heating rate of10° C. from ambient temperature to 300-350° C. The balance and furnacechambers were purged with nitrogen flow during use.

A representative TGA trace of the Form 1 crystalline free form of theinvention is shown in FIG. 8. The thermal gravimetric analysis (TGA)trace of FIG. 8 shows no significant weight loss at temperatures belowthe onset of decomposition at about 293° C.

A representative TGA trace of the Form 2 crystalline free form of theinvention is shown in FIG. 3. The thermal gravimetric analysis (TGA)trace of FIG. 3 shows no significant weight loss at temperatures belowthe onset of decomposition at about 269° C.

Example 8: Dynamic Moisture Sorption Assessment

Dynamic moisture sorption (DMS) measurement was performed using a VTIatmospheric microbalance, SGA-100 system (VTI Corp., Hialeah, Fla.33016). A weighed sample was used and the humidity was lowest possiblevalue (close to 0% RH) at the start of the analysis. The DMS analysisconsisted of an initial drying step (0% RH) for 120 minutes, followed bytwo cycles of sorption and desorption with a scan rate of 5% RH/stepover the humidity range of 5% RH to 90% RH. The DMS run was performedisothermally at 25° C.

A representative DMS trace for the Form 1 crystalline free form of theinvention is shown in FIG. 9.

Crystalline Form 1 demonstrated a small hysteresis between two cycles ofsorption and desorption. Form 1 demonstrated about 0.99% weight gain inthe humidity range of 5% to 70% relative humidity and about 1.32% weightgain in the humidity range of 5% to 90% relative humidity at roomtemperature, as shown in FIG. 9. Form 1 is considered to beslightly-hygroscopic.

A representative DMS trace for the Form 2 crystalline free form of theinvention is shown in FIG. 4. Crystalline Form 2 showed no hysteresisbetween two cycles of sorption and desorption and demonstrated anexceptionally small propensity for hygroscopicity. Form 2 demonstratedabout 0.12% weight gain in the humidity range of 5% to 70% relativehumidity and about 0.18° % weight gain in the humidity range of 5% to90% relative humidity at room temperature, as shown in FIG. 4. Form 2 isconsidered to be non-hygroscopic.

Example 9: Single Crystal X-Ray Diffraction of Form 2

Data were collected on a Rigaku Oxford Diffraction Supernova DualSource, Cu at Zero, Atlas CCD diffractometer equipped with an OxfordCryosystems Cobra cooling device. The data were collected using Cu Kαradiation. The structure was solved and refined using the Bruker AXSSHELXTL suite crystallographic software. Full details can be found inthe CIF. Unless otherwise stated, hydrogen atoms attached to carbon wereplaced geometrically and allowed to refine with a riding isotropicdisplacement parameter. Hydrogen atoms attached to the heteroatoms werelocated in a difference Fourier map and were allowed to refine freelywith an isotropic displacement parameter.

TABLE 3 Data from Single Crystal X-ray Diffraction Analysis for Form 2Empirical formula C₂₇H₂₈F₂N₆O₃ Formula weight  522.55 Crystal size 0.14× 0.10 × 0.02 mm³ Temperature of Data Collection 293(2) K Wavelengthused for Data Collection 1.54178 Å Crystal system Orthorhombic Spacegroup Pbca Unit cell dimensions a = 9.7245(11) Å b = 16.8197(14) Å c =32.604(4) Å α = 90° β = 90° γ = 90° Unit cell volume 5332.8(10) Å³ Z(Number of molecules in the unit   8 cell) Density (calculated) 1.302g/cm³ Theta range for data collection 5.26-66.60° Index ranges −11 ≤ h ≤11 −12 ≤ k ≤ 20 −38 ≤ l ≤ 38 Reflections collected 24516 Independentreflections 4708 [R_(int) = 0.0927] Final R indices [F2 > 2sigma(F2)] R1= 0.0808, wR2 = 0.2159 R indices (all data) R1 = 0.1452, wR2 = 0.2859

Example 10: Solid State Stability Assessment of Form 1

Samples of the Form 1 crystalline free form of the invention were storedat 25° C. and 60% relative humidity (RH) and at 40° C. and 75% RH undertwo configurations a) open glass vial and b) closed glass vial placedinside an HDPE bottle containing desiccant. HDPE bottle was inductionsealed. At specific intervals, the contents of a representative samplewere removed and analyzed by HPLC for chemical purity (shown below asHPLC Purity (% a/a)).

TABLE 4 Crystalline Form 1 Stability Study Condition 40° C./ 25° C/ Time75% RH 40° C./ 60% RH 25° C./ Point Closed With 75% RH Closed 60% RH(weeks) Desiccant Open With Desiccant Open 0 98.73 2 NT 98.72 NT NT 498.75 98.70 98.75 98.75 8 98.80 98.97 98.87 98.75 24 99.06 98.89 99.0298.78 36 98.86 98.71 98.80 98.75 NT: Not Tested

Example 11: Polarized Light Microscopy (PLM) Image of Form 1 and Form 2

Samples of Form 1 and Form 2 were examined under an optical microscope(Olympus BX51) with cross-polarized light filter. Images were collectedwith a PaxCam camera controlled by PaxIt Imaging Software (version 6.4).Samples were prepared on glass slides with light mineral oil asimmersion medium. Depending on the size of the particles, a 4×, a 10× ora 20× objective lens was used for magnification.

Preparation 8: tert-butyl2-iodo-1-((2-(trimethylsilyl)ethoxy)methyl)-1,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridine-5-carboxylate(C-5)

To a solution of compound C-22 (25 g, 135.86 mmol) in 0.01M HCl (500 mL)was added dimethoxymethane (21.64 mL, 244.54 mmol). The resultingsolution was stirred at 100° C. for 18 h. The solvent was removed undervacuum and the residue was triturated twice with diethyl ether andethanol, filtered and dried to provide compound C-21 (25.2 g, 94.6%yield).

To a solution of compound C-21 (25.0 g, 127.55 mmol) in methanol (250mL) was added DIPEA (57.23 mL, 318.87 mmol) followed by addition of(Boc)₂O (68.23 mL, 318.87 mol) and the reaction was stirred at RT for 18h. The resulting reaction was diluted with water (150 mL) and extractedusing ethyl acetate (3×200 mL). The combined organic layer was driedover anhydrous sodium sulfate, decanted and concentrated under reducedpressure to obtain crude product C-20 which was taken forward to thenext step without further purification.

To a solution of this crude compound C-20 (40.0 g, 123.8 mmol) inMethanol (500 mL) was added 1M NaOH Solution (200 mL) and resultingsolution was stirred at RT for 18 h. The solvent was distilled off undervacuum and the resulting crude diluted with water (200 mL) and extractedusing ethyl acetate (3×300 mL). The combined organic layer was driedover anhydrous sodium sulphate and concentrated under reduced pressureto obtain crude product which was purified through column chromatography(100-200 silica gel), eluted with 5-10% MeOH: DCM to get desired productC-19 (20 g, 72.4% yield).

To a solution of compound C-19 (20 g, 89.68 mmol) in THF (400 mL) wasadded NIS (30.26 g, 134.52 mmol) at RT and the resulting solution wasstirred for 2 hours at the same temperature. The reaction mixture wasdiluted with water (200 mL) and extracted in ethyl acetate (2×300 mL),the organic layer was washed with a 10% sodium thiosulphate aqueoussolution (3×100 mL) followed by brine, dried over anhydrous sodiumsulphate and concentrated under reduced pressure to obtain crude productC-18 which was used in the next step without further purification.

To a stirred solution of compound C-18 (15.0 g, 42.97 mmol) in THF (150mL) was added NaH (1.8 g 45.11 mmol) at 0° C. portionwise and thereaction mixture was stirred for 1 hour at RT. Then SEM chloride (8.18mL, 45.11 mmol) was added dropwise at 0° C. The reaction was stirred for6 hours at RT. Progress of reaction was monitored by TLC, the reactionwas quenched with ice water (200 mL) at 0° C. and extracted with ethylacetate (2×200 mL). The combined organic layer was washed with brine anddried over anhydrous sodium sulphate. The organic layer was filtered andconcentrated under reduced pressure to obtain crude product, which waspurified by column chromatography (100-200) silica (eluted with 10-15%EtOAc: Hexane) to get the desired product C-5 as a viscous liquid (11 g,55%).

Preparation 9:2-(2-ethyl-5-fluoro-4-methoxyphenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane(C-12)

To a stirred suspension of compound C-17 (347.6 g, 973.14 mmol) inanhydrous THF (1000 mL) cooled to −40° C., was added n-Butyl lithium(2.5 M in hexane, 362.6 mL, 905.02 mmol) over 50 min, at which point thecharacteristic yellow color of the phosphorus ylide persisted. Thereaction mixture was warmed to −10° C. and stirred for 1 h then themixture was cooled to −30° C. and a solution of compound R-1 (50 g,324.38 mmol) in anhydrous THF (200 mL) was added over 30 min. Theresultant mixture was warmed to ambient temperature and stirredovernight. Progress of the reaction was monitored by TLC. On completion,the reaction was quenched by gradual addition of water (500 mL) andextracted with diethyl ether (3×500 mL). The combined organic layer waswashed with water (2×500 mL), brine (250 mL), dried over (anhydrousNa₂SO₄) and concentrated under reduced pressure to give crude compound.The obtained crude product C-16 was used in the next step withoutpurification.

To a solution crude C-16 (110 g, 723.39 mmol) in ethanol (1000 mL) wasadded 10% Pd/C (50 g). A balloon of hydrogen gas was mounted and thereaction was evacuated and back-filled with hydrogen three times. Thereaction was stirred under a hydrogen atmosphere overnight at roomtemperature. After stirring at RT overnight, the reaction was complete.It was filtered through a pad of Celite and concentrated in vacuo toprovide crude compound, which was purified through column chromatography(100-200) silica gel, eluted using 3-5% ethyl acetate/hexane to obtainthe desired product C-15 as colorless liquid (24 g, 48% over the 2steps).

To a solution of compound C-15 (24.0 g, 155.84 mmol) in MeCN (200 mL)was added a solution of NBS (28.0 g, 157.40 mmol) in MeCN (100 mL). Theresulting solution was stirred at room temperature for 18 h. Solvent wasremoved in vacuo and the residue was diluted with diethyl ether (100mL). Precipitation observed, which was removed by filtration and thefiltrate was washed with sodium sulfite aqueous solution (200 mL) andbrine (100 mL), dried over anhydrous sodium sulphate and concentrated invacuo to give the desired product C-14 as yellow oil (35.0 g, 97%yield).

To a solution of compound C-14 (20 g, 85.836 mmol) in Dioxane (400 mL)were added compound R-2 (32.69 g, 128.755 mmol) and KOAc (25.27 g,257.508 mmol). The reaction mixture was degassed with nitrogen for 15minutes then palladium catalyst (3.5 g, 4.29 mmol) was added. Thereaction mixture was stirred and heated at 110° C. for 3 hours. Thereaction was filtered through a pad of Celite and washed with ethylacetate. The filtrate was diluted with ethyl acetate (200 mL) and washedwith water (2×200 mL)) and brine (100 mL), dried over sodium sulphateand concentrated under vacuum. The crude product obtained was purifiedthrough column chromatography (100-200 silica gel), eluted with 3-5%EtOAc: Hexane to give the desired product C-12 (20 g, 83% yield).

Preparation 10:3-(dimethyl-stannyl)-6-(2-ethyl-5-fluoro-4-methoxyphenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazole(C-6)

A mixture of compound C-13 (25 g, 126.84 mmol), 3,4-dihydro-2H-pyran(134.5 mL, 1471.5 mL) and p-TSA (5.57 g, 29.18 mmol) was taken in THF(700 mL) and heated at 60° C. overnight. The reaction mixture was pouredinto ice water and the aqueous phase was extracted with ethyl acetate.The organic layer was dried over sodium sulfate and filtered. Thefiltrate was evaporated under reduced pressure and residue purified oversilica gel (230-400) column (eluting with 1-2% ethyl acetate in hexane)to give desired compound C-11 (23.5 g, 67% yield).

A solution of compound C-11 (13.3 g, 47.5 mmol), compound C-12 (15.96 g,57.0 mmol) and K₃PO₄ (30.21 g, 142.5 mmol) in DMF:H₂O (396:99 mL) wasdegassed with nitrogen for 15 minutes then palladium catalyst (1.6 g,2.37 mmol) was added and the reaction mixture was purged with nitrogenfor 5 minutes. The resulting reaction mixture was heated at 100° C. for12 h under continuous stirring. The reaction mixture was filteredthrough a pad of Celite and washed with ethyl acetate. The filtrate wasdiluted with ethyl acetate (200 mL), extracted with EtOAc (2×100 mL) andwashed with cold water (100 mL) and brine (50 mL), dried over sodiumsulphate and concentrated under vacuum to get crude product which waspurified through flash chromatography (100-200 silica gel), eluted with10% EtOAc:Hexane to give C-10 (14 g, 91.4% yield)).

To a solution of compound C-10 (52 g, 146.89 mmol) in methanol (600 mL)was added Concentrated HCl (50 mL) and the resulting solution was heatedat 60° C. overnight. The reaction mixture was cooled to RT andconcentrated under vacuum. The residue was diluted with EtOAc (200 mL)and washed with a saturated NaHCO₃ aqueous solution (2×150 mL). Theorganic layer was dried over Na₂SO₄ and concentrated to get the desiredproduct C-9 (35 g, 88.9% yield).

To a solution of compound C-9 (17.5 g, 64.81 mmol) in DMF (100 mL) wasadded KOH (14.5 g, 259.54 mmol) and the mixture was stirred for 15minutes. A solution of iodine (32.7 g, 129.62 mmol) in DMF (50 mL) wasadded slowly at 0° C. and the reaction mixture was stirred at RT for 30min. Progress of the reaction was monitored by TLC then the reactionmixture was diluted with water (200 mL) and extracted with ethyl acetate(2×200 mL). The organic layer was washed with saturated sodiummetabisulfite aqueous solution (2×150 mL) and water (3×100 mL), driedover Na₂SO₄ and concentrated under vacuum to get crude product C-8 (21g).

To a solution of C-8 (21.0 g, 53.02 mmol) in DCM (230 mL) was addedp-TsOH (1.8 g, 10.60 mmol) and the mixture was cooled to 0° C. CompoundR-3 (7.04 mL, 79.54 mmol) was added drop wise to the above solution andthe reaction mixture was stirred at RT overnight. TLC monitoring showedcompletion of the reaction. The reaction mixture was diluted with DCM(2×150 mL) and washed with saturated NaHCO₃ aqueous solution (200 mL)and brine (200 mL). The organic layer was dried over anhydrous Na₂SO₄and concentrated in vacuo to give crude product which was purified byflash chromatography to give C-7.

A solution of C-7 (10.0 g, 20.83 mmol) in Toluene (200 mL) was degassedwith nitrogen for 20 minutes followed by addition of R-4 (4.89 mL, 22.91mmol) and Pd(PPh₃)₄ (1.2 g, 1.04 mmol). The reaction mixture was purgedwith nitrogen for an additional 5 minutes and then stirred at 100° C.After 2 h, TLC shows completion of the reaction. The reaction mixturewas cooled to room temperature, filtered through a Celite pad and theresidue was washed with ethyl acetate. The concentrated filtrate waspurified by column chromatography (neutral alumina), eluted with 2-5%EtOAc: Hexane to give product C-6 (6.4 g, 57.6% yield).

Preparation 11:2-(6-(2-ethyl-5-fluoro-4-methoxyphenyl)-1H-indazol-3-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine(C-3)

To a solution of compound C-6 (6.4 g, 12.37 mmol) in Toluene (100 mL)was added Compound C-5 (5.9 g, 12.37 mmol). The reaction mixture wasdegassed with nitrogen for 20 minutes, followed by addition of copper(I) iodide (470 mg, 2.47 mmol) and Pd(PPh₃)₄ (714 mg, 1.237 mmol) thenstirred at 100° C. for 12 h. Progress of the reaction was monitored byTLC. The reaction was cooled to RT and filtered through a Celite pad,the residue was washed with ethyl acetate. The organic layer was dilutedwith water, separated and the organic part was washed with brine, driedover Na₂SO₄ and filtered. The filtrate was concentrated under reducedpressure to give crude product which was purified by columnchromatography (100-200 mesh size silica), eluted with 20%, EtOAc:Hexane to give C-4 (4 g, 45.90/%).

To a solution of compound C-4 (4.0 g, 5.6 mmol) in Dioxane (30 mL) wasadded concentrated HCl (30 mL). The reaction mixture was stirred at 70°C. for 16 h. Progress of the reaction was monitored by LCMS. Thereaction was cooled to RT, concentrated in vacuo, triturated withdiethyl ether and purified through prep HPLC to give the desiredcompound C-3 (0.65 g, 29.5%).

Example 12:1-(2-(6-(2-Ethyl-5-fluoro-4-hydroxyphenyl)-1H-indazol-3-yl)-1,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridin-5-yl)-2-morpholinoethan-1-one(C-1)

To the mixture of C-3 (180 mg, 0.460 mmol) in DCM (0.5 ml) at rt wasadded boron tribromide, 1 m in DCM [100 ml) (2.299 ml, 2.299 mmol). Theresulting mixture was stirred for 30 mins before it was concentrated.The resulting residue was co-evaporated with MeOH (3×3.0 mL),re-dissolved in 1:1 mixture of AcOH:H₂O (3.0 mL), filtered and purifiedby reverse phase prep HPLC. Desired fractions were combined and frozendried to give C-2((5-ethyl-2-fluoro-4-(3-(4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)phenol)as a TFA salt.

To the mixture of C-2, TFA (15 mg, 0.031 mmol) and 7″ (2 equivalents,0.061 mmol) in DMF (0.5 ml) at rt was added HATU (25.5 mg, 0.067 mmol)and DIEA (0.043 ml, 0.244 mmol). The resulting mixture was stirred at rtovernight. The reaction was diluted with MeOH (0.5000 ml) and water(0.500 ml). LiOH (2.193 mg, 0.092 mmol) was added. The resulting mixturewas heated at 65° C. for 1 hr. The reaction was then concentrated, theresulting residue was treated with a mixture of DCM (0.500 ml) and TFA(0.500 ml) at rt for 30 mins, concentrated, re-dissolved in 1:1 mixtureof AcOH:H₂O (1.5 mL), filtered and purified by reverse phase prep HPLCto give compound C-1 as a TFA salt. (m/z): [M+H]⁺ calcd for C₂₇H₂₉FN₆O₃504.23 found 505.2.

Biological Assays

Compound 1 has been characterized in one or more of the followingbiological assays.

Assay 1: Biochemical JAK Kinase Assays

A panel of four LanthaScreen JAK biochemical assays (JAK1, 2, 3 andTyk2) were carried in a common kinase reaction buffer (50 mM HEPES, pH7.5, 0.01% Brij-35, 10 mM MgCl₂, and 1 mM EGTA). Recombinant GST-taggedJAK enzymes and a GFP-tagged STAT1 peptide substrate were obtained fromLife Technologies.

Serially diluted compound was pre-incubated with each of the four JAKenzymes and the substrate in white 384-well microplates (Corning) atambient temperature for 1 h. ATP was subsequently added to initiate thekinase reactions in 10 μL total volume, with 1% DMSO. The final enzymeconcentrations for JAK1, 2, 3 and Tyk2 are 4.2 nM, 0.1 nM, 1 nM, and0.25 nM respectively; the corresponding Km ATP concentrations used are25 μM, 3 μM, 1.6 μM, and 10 μM; while the substrate concentration is 200nM for all four assays. Kinase reactions were allowed to proceed for 1hour at ambient temperature before a 10 μL preparation of EDTA (10 mMfinal concentration) and Tb-anti-pSTAT1 (pTyr701) antibody (LifeTechnologies, 2 nM final concentration) in TR-FRET dilution buffer (LifeTechnologies) was added. The plates were allowed to incubate at ambienttemperature for 1 h before being read on the EnVision reader (PerkinElmer). Emission ratio signals (520 nm/495 nm) were recorded andutilized to calculate the percent inhibition values based on DMSO andbackground controls.

For dose-response analysis, percent inhibition data were plotted vs.compound concentrations, and IC₅₀ values were determined from a4-parameter robust fit model with the Prism software (GraphPadSoftware). Results were expressed as pIC₅₀ (negative logarithm of IC₅₀)and subsequently converted to pK_(i)(negative logarithm of dissociationconstant, Ki) using the Cheng-Prusoff equation.

Compound 1 exhibited the following enzyme potency.

TABLE 5 JAK 1 JAK 2 JAK 3 Tyk2 pK_(i) pK_(i) pK_(i) Pk_(i) 10 10.6 9.78.7

Assay 2: Cellular JAK Potency Assay: Inhibition of IL-13

The AlphaScreen JAKI cellular potency assay was carried out by measuringinterleukin-13 (IL-13, R&D Systems) induced STAT6 phosphorylation inBEAS-2B human lung epithelial cells (ATCC). The anti-STAT6 antibody(Cell Signaling Technologies) was conjugated to AlphaScreen acceptorbeads (Perkin Elmer), while the anti-pSTAT6 (pTyr641) antibody (CellSignaling Technologies) was biotinylated using EZ-Link Sulfo-NHS-Biotin(Thermo Scientific).

BEAS-2B cells were grown at 37° C. in a 5% CO₂ humidified incubator in50% DMEM/50% F-12 medium (Life Technologies) supplemented with 10% FBS(Hyclone), 100 U/mL penicillin, 100 μg/mL streptomycin (LifeTechnologies), and 2 mM GlutaMAX (Life Technologies). On day 1 of theassay, cells were seeded at a 7,500 cells/well density in whitepoly-D-lysine-coated 384-well plates (Corning) with 25 μL medium, andwere allowed to adhere overnight in the incubator. On day 2 of theassay, the medium was removed and replaced with 12 μL of assay buffer(Hank's Balanced Salt Solution/HBSS, 25 mM HEPES, and 1 mg/mL bovineserum albumin/BSA) containing dose-responses of test compounds. Thecompound was serially diluted in DMSO and then diluted another 1000-foldin media to bring the final DMSO concentration to 0.1%. Cells wereincubated with test compounds at 37° C. for 1 h, and followed by theaddition of 12 μl of pre-warmed IL-13 (80 ng/mL in assay buffer) forstimulation. After incubating at 37° C. for 30 min, the assay buffer(containing compound and IL-13) was removed, and 10 μL of cell lysisbuffer (25 mM HEPES, 0.1% SDS, 1% NP-40, 5 mM MgCl₂, 1.3 mM EDTA, 1 mMEGTA, and supplement with Complete Ultra mini protease inhibitors andPhosSTOP from Roche Diagnostics). The plates were shaken at ambienttemperature for 30 min before the addition of detection reagents. Amixture of biotin-anti-pSTAT6 and anti-STAT6 conjugated acceptor beadswas added first and incubated at ambient temperature for 2 h, followedby the addition of streptavidin conjugated donor beads (Perkin Elmer).After a minimum of 2 h incubation, the assay plates were read on theEnVision plate reader. AlphaScreen luminescence signals were recordedand utilized to calculate the percent inhibition values based on DMSOand background controls.

For dose-response analysis, percent inhibition data were plotted vs.compound concentrations, and IC₅₀ values were determined from a4-parameter robust fit model with the Prism software. Results may alsobe expressed as the negative logarithm of the IC₅₀ value, pIC₅₀.Compound 1 exhibited a pIC₅₀ value of 7.9 in this assay.

Assay 3: Cellular JAK Potency Assay: Inhibition of IL-2/Anti-CD3Stimulated IFNγ in Human PBMCs

The potency of the test compound for inhibition of interleukin-2(IL-2)/anti-CD3 stimulated interferon gamma (IFNγ) was measured in humanperipheral blood mononuclear cells (PBMCs) isolated from human wholeblood (Stanford Blood Center). Because IL-2 signals through JAK, thisassay provides a measure of JAK cellular potency.

(1) Human peripheral blood mononuclear cells (PBMC) were isolated fromhuman whole blood of healthy donors using a ficoll gradient. Cells werecultured in a 37° C., 5% CO₂ humidified incubator in RPMI (LifeTechnologies) supplemented with 10% Heat Inactivated Fetal Bovine Serum(FBS, Life Technologies), 2 mM Glutamax (Life Technologies), 25 mM HEPES(Life Technologies) and 1× Pen/Strep (Life Technologies). Cells wereseeded at 200,000 cells/well in media (50 μL) and cultured for 1 h. Thecompound was serially diluted in DMSO and then diluted another 500-fold(to a 2× final assay concentration) in media. The test compounddilutions (100 μL/well) were added to cells, and incubated at 37° C., 5%CO₂ for 1 h, followed by the addition of IL-2 (R&D Systems; finalconcentration 100 ng/mL) and anti-CD3 (BD Biosciences; finalconcentration 1 μg/mL) in pre-warmed assay media (50 μL) for 24 h.

(2) After cytokine stimulation, cells were centrifuged at 500 g for 5min and supernatants removed and frozen at −80° C. To determine theinhibitory potency of test compounds in response to IL-2/anti-CD3,supernatant IFNγ concentrations were measured via ELISA (R&D Systems).IC₅₀ values were determined from analysis of the inhibition curves ofconcentration of IFNγ vs compound concentration. Data are expressed aspIC₅₀ (negative decadic logarithm IC₅₀) values. Compound 1 exhibited apIC₅₀ value of about 6.7 in this assay.

Assay 4: Cellular JAK Potency Assay: Inhibition of IL-2 StimulatedpSTAT5 in CD4+ T Cells

The potency of the test compound for inhibition of interleukin-2(IL-2)/anti-CD3 stimulated STAT5 phosphorylation was measured inCD4-positive (CD4+) T cells in human peripheral blood mononuclear cells(PBMCs) isolated from human whole blood (Stanford Blood Center) usingflow cytometry. Because IL-2 signals through JAK, this assay provides ameasure of JAK cellular potency.

CD4+ T cells were identified using a phycoerythrobilin (PE) conjugatedanti-CD4 antibody (Clone RPA-T4, BD Biosciences), while an Alexa Fluor647 conjugated anti-pSTAT5 antibody (pY694, Clone 47, BD Biosciences)was used to detect STAT5 phosphorylation.

(1) The protocol of Assay 3 paragraph (1) was followed with theexception that the cytokine stimulation with IL-2/anti-CD3 was performedfor 30 min instead of 24 h.

(2) After cytokine stimulation, cells were fixed with pre warmed fixsolution (200 μL; BD Biosciences) for 10 min at 37° C., 5% CO₂, washedtwice with DPBS buffer (1 mL, Life Technologies), and resuspended in icecold Perm Buffer III (1000 μL, BD Biosciences) for 30 min at 4° C. Cellswere washed twice with 2% FBS in DPBS (FACS buffer), and thenresuspended in FACS buffer (100 μL) containing anti-CD4 PE (1:50dilution) and anti-CD3 anti-CD3Alexa Fluor 647 (1:5 dilution) for 60 minat room temperature in the dark. After incubation, cells were washedtwice in FACS buffer before being analyzed using a LSRII flow cytometer(BD Biosciences). To determine the inhibitory potency of the testcompound in response to IL-2/anti-CD3, the median fluorescent intensity(MFI) of pSTAT5 was measured in CD4+ T cells. IC₅₀ values weredetermined from analysis of the inhibition curves of MFI vs compoundconcentration. Data are expressed as pIC₅₀ (negative decadic logarithmIC₅₀) values. Compound 1 exhibited a pIC₅₀ value of about 7.7 in thisassay.

Assay 5: Cellular JAK Potency Assay: Inhibition of IL-6 Stimulated CCL2(MCP-1) in Human PBMCs

The potency of the test compound for inhibition of interleukin-6 (IL-6)stimulated CCL2 (MCP-1) production was measured in human peripheralblood mononuclear cells (PBMCs) isolated from human whole blood(Stanford Blood Center). Because IL-6 signals through JAK, this assayprovides a distal measure of JAK cellular potency.

(1) The protocol of Assay 3 paragraph (1) was followed up to theincubation with test compounds. In the present assay, after testcompounds were added to wells and incubated, IL-6 (R&D Systems; finalconcentration 10 ng/ml) in pre-warmed assay media (50 μL) was added.

(2) After cytokine stimulation for 48 h, cells were centrifuged at 500 gfor 5 min and supernatants were removed and frozen at −80° C. Todetermine the inhibitory potency of the test compound in response toIL-6, supernatant CCL2 (MCP-1) concentrations were measured via ELISA(R&D Systems). IC₅₀ values were determined from analysis of theinhibition curves of concentration of CCL2/MCP-1 vs compoundconcentration. Data are expressed as pIC₅₀ (negative decadic logarithmIC₅₀) values. Compound 1 exhibited a pIC₅₀ value of about 6.4 in thisassay.

Assay 6: Cellular JAK Potency Assay: Inhibition of IFNγ-Induced pSTAT1

The potency of the test compound for inhibition of interferon gamma(IFNγ) stimulated STAT1 phosphorylation was measured in CD14-positive(CD14+) monocytes derived from human whole blood (Stanford Blood Center)using flow cytometry. Because IFNγ signals through JAK, this assayprovides a measure of JAK cellular potency.

Monocytes were identified using a fluorescein isothiocyanate (FITC)conjugated anti-CD14 antibody (Clone RM052, Beckman Coulter), and anAlexa Fluor 647 conjugated anti-pSTAT1 antibody (pY701, Clone 4a, BDBiosciences) was used to detect STAT1 phosphorylation.

Human peripheral blood mononuclear cells (PBMC) were isolated from humanwhole blood of healthy donors using a ficoll gradient. Cells werecultured in a 37° C., 5% CO₂ humidified incubator in RPMI (LifeTechnologies) supplemented with 10% Fetal Bovine Serum (FBS, LifeTechnologies), 2 mM Glutamax (Life Technologies), 25 mM HEPES (LifeTechnologies) and 1× Pen/Strep (Life Technologies). Cells were seeded at250,000 cells/well in media (200 μL), cultured for 2 h and resuspendedin assay media (50 μL) (RPMI supplemented with 0.1% bovine serum albumin(Sigma), 2 mM Glutamax, 25 mM HEPES and 1× Penstrep) containing variousconcentrations of test compounds. Compounds were serially diluted inDMSO and then diluted another 1000-fold in media to bring the final DMSOconcentration to 0.1%. The test compound dilutions were incubated withcells at 37° C., 5% CO₂ for 1 h, followed by the addition of pre-warmedIFNγ (R&D Systems) in media (50 μL) at a final concentration of 0.6ng/mL for 30 min. After cytokine stimulation, cells were fixed withpre-warmed fix solution (100 μL) (BD Biosciences) for 10 min at 37° C.,5% CO₂, washed twice with FACS buffer (1 mL) (1% BSA in PBS),resuspended in 1:10 anti-CD14 FITC:FACS buffer (100 μL), and incubatedat 4° C. for 15 min. Cells were washed once, and then resuspended in icecold Perm Buffer III (BD Biosciences) (100 μL) for 30 min at 4° C. Cellswere washed twice with FACS buffer, and then resuspended in 1:10anti-pSTAT1 Alexa Fluor 647:FACS buffer (100 μL) for 30 min at RT in thedark, washed twice in FACS buffer, and analyzed using a LSRII flowcytometer (BD Biosciences).

To determine the inhibitory potency of the test compound, the medianfluorescent intensity (MFI) of pSTAT1 was measured in CD14+ monocytes.IC₅₀ values were determined from analysis of the inhibition curves ofMFI vs compound concentration. Data are expressed as pIC₅₀ (negativedecadic logarithm IC₅₀) values. Compound 1 exhibited a pIC₅₀ value ofabout 7.1 in this assay.

Assay 7: Ocular Pharmacokinetics in Rabbit Eyes

The objective of this assay was to determine the pharmacokinetics of atest compound in rabbit ocular tissues.

Solution Formulation

1-(2-(6-(2-ethyl-5-fluoro-4-hydroxyphenyl)-4-fluoro-1H-indazol-3-yl)-1,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridin-5-yl)-2-morpholinoethan-1-one(1), prepared in Example 2, was dissolved in 2%2-hydroxypropyl-β-cyclodextrin to attain a target concentration of 1mg/mL. Bilateral intravitreal injection (50 μL/eye) of the solution oftest compound was administered to New Zealand white rabbits (50 μg/eye).The test compound concentration was measured in ocular tissues:vitreous, aqueous, retina/choroid and iris-ciliary body atpre-determined time points post injection (30 min, 4 h, 1 d, 3 d, 7 d,14 d). Two rabbits (four eyes) were dosed for each time point. In thevitreous tissue, compound 1 exhibited a two-phase decrease inconcentration characterized by an initial decrease in concentration witha half-life of approximately 9 hours and finally a terminal half-life ofapproximately 2 days. The compound was found to distribute quickly intothe retinal and choroidal region as well and shows a similarpharmacokinetic profile as in the vitreous tissue.

Suspension Formulation

A suspension formulation was prepared by combining compound 1 of Example2 (Form 1), with 0.5% hydroxypropyl methylcellulose (HPMC E5)+0.02%Tween 80 in normal saline to attain a target concentration of 5 mg/mL,20 mg/mL and 80 mg/mL for the 0.25 mg/eye, 1 mg/eye and 4 mg/eye dosesrespectively. Bilateral intravitreal injection (50 μL/eye) of thesuspension of test compound was administered to New Zealand whiterabbits. The test compound concentration was measured in ocular tissuesas in the solution formulation assay at 30 min, 4 h, 24 h, 72 h, 7 d, 14d, 28 d, 56 d and 84 d post injection. For the 4 mg/eye dose group, anadditional time point at 168 d post injection was also collected. Alldose groups demonstrated measurable drug concentration in the eye up tothe last time point tested in this study. Robust sustained exposure wasobserved for all doses at 12 weeks (84 d). Sustained exposure wasobserved at 24 weeks (84 d) for the 4 mg/eye does group. The compoundshowed a linear decrease in drug concentration in the vitreous tissuefrom 30 min to 24 weeks with a drug clearance rate of approximately 5 to10 μg/mL/day. The clearance rate is consistent with the solubility ofcompound 1 in the vehicle and the ocular pharmacokinetic behavior in thesolution formulation. All dose groups demonstrated measurable drugconcentration in the eye up to the last time point tested in this study.Therefore, it is plausible that drug exposure is longer than thatobserved in this study. The drug concentration in plasma was measuredand found to be at least 3 orders of magnitude lower than theconcentration in vitreous tissue at all three concentrations.

A suspension formulation was prepared by combining compound 1 of Example5 (Form 2), with 0.5% hydroxypropyl methylcellulose (HPMC E5)+0.02%Tween 80 in normal saline to attain a target concentration of 0.4 mg/mL,1 mg/mL, 2 mg/mL and 20 mg/mL for the 0.02 mg/eye, 0.05 mg/eye, 0.1mg/eye and 1 mg/eye doses respectively. Bilateral intravitreal injection(50 μL/eye) of the suspension of test compound was administered to DutchBelted rabbits. The test compound concentration was measured in vitreoushumor, aqueous humor, iris-ciliary body, retina, retinal pigmentepithelial/choroidal cells and plasma at 30 min, 7 d, 14 d, 28 d, 42 dand 56 d post injection. The compound showed a very gradual decrease indrug concentration in the vitreous tissue from 30 min to the last timepoint tested (28 d for 0.02 mg/eye dose, 42 d for 0.05 mg/eye and 1mg/eye doses and 56 d for the 0.1 mg/eye dose). All dose groupsdemonstrated measurable drug concentration in the eye up to the lasttime point tested in this study. Therefore, it is plausible that drugexposure is longer than that observed in this study. The drugconcentration in plasma was measured and found to be at least 3 ordersof magnitude lower than the concentration in vitreous tissue at allthree concentrations. The 3 orders of magnitude correspond to alogarithmic scale (ie 1000 on a non-logarithmic scale).

Assay 8: Pharmacodynamic Assay: Inhibition of IL6-Induced pSTAT3 in Rats

The ability of a single intravitreal administration of test compound toinhibit IL-6 induced pSTAT3 was measured in rat retina/choroidhomogenates.

A suspension formulation was prepared by combining compound 1 of Example2, with 0.5% hydroxypropyl methylcellulose (HPMC E5 LV), 0.02% Tween 80,and 9 mg/mL sodium chloride in purified water to attain a targetconcentration of 10 mg/mL.

Female Lewis rats were intravitreally (IVT) dosed (5 μL per eye) withthe suspension formulation. Three days later, IL-6 (Peprotech; 0.1mg/mL; 5 μL per eye) or vehicle was intravitreally administered toinduce pSTAT3. Ocular tissues were dissected one hour after the secondIVT injection with IL-6. The retina/choroid tissues were homogenized andpSTAT3 levels were measured using an ELISA (Cell Signaling Technology).The percent inhibition of IL-6-induced pSTAT3 was calculated incomparison to the vehicle/vehicle and vehicle/IL-6 groups. Inhibition ofgreater than 100% reflects a reduction of pSTAT3 levels to below thoseobserved in the vehicle/vehicle group.

With a 3 day pre-treatment prior to IL-6 challenge, a 50 μg dose ofcompound 1 administered by the suspension formulation inhibitedIL-6-induced pSTAT3 by 116% in the retina/choroid tissues.

Assay 9: Pharmacodynamic Assay: Inhibition of IFNγ-Induced IP-10 inRabbit Eyes

The ability of a single intravitreal administration of test compound toinhibit interferon-gamma (IFNγ) induced IP-10 protein levels wasmeasured in rabbit vitreous and retina/choroid tissues.

A suspension formulation was prepared by combining compound 1 of Example2 (Form 1), with 0.5% hydroxypropyl methylcellulose (HPMC E5), 0.02%Tween 80, and 9 mg/mL sodium chloride in purified water to attain atarget concentration of 20 mg/mL.

Male, New Zealand White rabbits (Liveon Biolabs, India) were used forthe studies. Animals were acclimated after arrival at the researchfacilities (Jubilant Biosys Ltd., India). Each rabbit was given a totalof two intravitreal (IVT) injections with a total dose volume of 50 μLper eye. The first IVT injection (45 μL per eye) delivered 0.9 mg oftest compound or vehicle. One week later, a second IVT injection (5 μLper eye) delivered IFNγ (1 μg/eye; stock solution 1 mg/mL; KingfisherBiotech) or vehicle for the induction of IP-10. On the day of theinjections, rabbits were anesthetized with an intramuscular injection ofketamine (35 mg/kg) and xylazine (5 mg/kg). Once deeply anesthetized,each eye was rinsed with sterile saline and IVT injections wereperformed using a 0.5 mL insulin syringe (50 units=0.5 mL) with a31-gauge needle at the supra-nasal side of the both eyes by marking theposition with a Braunstein fixed caliper (2¾″) 3.5 mm from the rectusmuscle and 4 mm from the limbus.

Tissues were collected 24 hours after the second IVT injection withIFNγ. Vitreous humor (VH) and retina/choroid tissues (R/C) werecollected and homogenized, and IP-10 protein levels were measured usinga rabbit CXCL10 (IP-10) ELISA kit (Kingfisher Biotech). The percentinhibition of IFNγ-induced IP-10 was calculated in comparison to thevehicle/vehicle and vehicle/IFNγ groups.

With a 1 week pre-treatment prior to the IFNγ challenge, the suspensionformulation of compound 1 inhibited IFNγ-induced IP-10 by 81% and 80% inthe vitreous humor and retina/choroid tissues, respectively. Similarefficacy was also observed with a 1 month pre-treatment prior to theIFNγ challenge.

Assay 10: Dermal Pharmacokinetics in Mouse and Mini-Pig Skin

The objective of this assay was to determine the epidermal, dermal andplasma pharmacokinetics of a test compound following a 24 hr exposure tointact mouse or mini-pig skin.

1-(2-(6-(2-Ethyl-5-fluoro-4-hydroxyphenyl)-4-fluoro-1H-indazol-3-yl)-1,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridin-5-yl)-2-morpholinoethan-1-one(1), was formulated to 0.5% (w/w) in cream or ointment as described, asFormulation A or Formulation B, respectively in Table 6.

Twenty-four hours prior to dosing the hair was shaved from the back of25 g male Balb/c mice exposing an area at of least 6 cm² (about 10% ofbody surface) and, in a separate experiment, of 10 kg Gottingenmini-pigs exposing an area of at least 450 cm² (about 10% of bodysurface). At time zero, following isoflurane anesthesia, the testcompound was applied to the back of mice or mini-pigs at a dose of 25μL/cm². The skin was covered with an adhesive cover to prevent loss ofcompound to the cage or bedding.

Following 24 h exposure, the backs were gently washed with soap andwater to remove non-absorbed drug and patted dry. Immediately followingthis washing, blood was drawn by cardiac puncture from the mice and viavenipuncture from the mini-pigs. The outer skin (stratum corneum) wasthen removed by adhesive tape stripping. Upon exposure of the epidermisa 0.5 cm punch biopsy was taken. The epidermis and dermis were quicklyseparated, weighed and snap frozen. Similar samples were obtained at 48h post dosing in mice and at 48 h, 94 h, and 168 h (7 days) post-dosingin mini-pigs.

Epidermis and dermis samples were homogenized in 1:10 (w/v) water usinga Covaris ultrasonic homogenizer. Samples were extracted in 3 volumes ofacetonitrile and quantified against a standard curve via LC-MS analysis.As evidenced by the pharmacokinetic parameters AUC_(0-t), for plasma,epidermis and dermis shown in Table 7 below, significant compoundexposure was exhibited in epidermis and dermis layers while the plasmaexposure was negligible in mice in Formulation A and below the limit ofquantitation in Formulation B in mice and in both formulations inmini-pig.

TABLE 6 Formulation A Formulation B Compound 1 0.5%   Compound 1 0.5%  Stearic Acid 5% Octylhydroxystearate 5% Cetostearyl Alcohol 5% C8-C10Triglyceride 5% Isopropyl Palmitate 4% Vaseline (Petrolatum) 79.5%  Octylhydroxystearate 2% N-Methylpyrrolidone 10%  BRIJ S2 1.08%   (PEG 2Stearyl Ether) BRIJ S20 6.92%   (PEG 20 Stearyl Ether)N-Methylpyrrolidine 10%  PEG400 10%  RO Water 55.5%  

TABLE 7 Plasma Epidermis Dermis AUC_(0-t) AUC_(0-t) AUC_(0-t) (μg *hr/mL) (μg * hr/g) (μg * hr/g) Mouse 0.022 1370 99 Formulation A Mouse<0.001 10700 1110 Formulation B Mini-pig <0.001 1220 44 Formulation AMini-pig <0.001 2460 88 Formulation B

Assay 11: Lung and Plasma Pharmacokinetics in Mice

Plasma and lung concentrations of compound 1 and ratios thereof weredetermined in the following manner. BALB/c mice from Charles RiverLaboratories were used in the assay. Compound 1 Form 1 of example 2 wasformulated in 0.01% Tween 80 in normal saline (0.9% sodium chloride inwater) at a concentration of 0.1 mg/mL as a suspension. 50 μL of thesuspension formulation was introduced into the trachea of a mouse byoral aspiration. At various time points (0.083, 1, 4, 24, 48, 72, and 96hr). Post dosing, blood samples were removed via cardiac puncture andintact lungs were excised from the mice. Blood samples were centrifuged(Eppendorf centrifuge, 5804R) for 4 minutes at approximately 12,000 rpmat 4° C. to collect plasma. Lungs were padded dry, weighed, andhomogenized at a dilution of 1:3 in sterile water. Plasma and lungconcentrations of compound 1 were determined by LC-MS analysis againstanalytical standards constructed into a standard curve in the testmatrix. Good exposure in lungs was found with a lung AUC (0-96 hr) of360 μg hr/g. The lung half-life was calculated at approximately 40hours. The lung to plasma ratio was determined as the ratio of the lungAUC in μg hr/g to the plasma AUC in μg hr/mL (where AUC isconventionally defined as the area under the curve of test compoundconcentration vs. time). The lung to plasma AUC ratio was 1780, showingvery low exposure in the plasma.

Assay 12: Pharmacodynamic Assay: Inhibition of IFNγ-Induced pSTAT1 inRabbit Eyes

The ability of a single intravitreal administration of test compound toinhibit interferon-gamma (IFNγ) induced phosphorylation of STAT1 protein(pSTAT1) was measured in rabbit retina/choroid tissue.

A suspension formulation was prepared by combining compound 1 of Example2 (Form 1), with 0.5% hydroxypropyl methylcellulose (HPMC E5), 0.02%Tween 80, and 9 mg/mL sodium chloride in purified water to attain atarget concentration of 20 mg/mL.

Male, New Zealand White rabbits (Liveon Biolabs, India) were used forthe studies. Animals were acclimated after arrival at the researchfacilities (Jubilant Biosys Ltd., India). Each rabbit was given a totalof two intravitreal (IVT) injections with a total dose volume of 50 μLper eye. The first IVT injection (45 μL per eye) delivered 0.9 mg oftest compound or vehicle. One week later, a second IVT injection (5 μLper eye) delivered IFNγ (1 μg/eye stock solution 1 mg/mL KingfisherBiotech) or vehicle for the induction of IP-10. On the day of theinjections, rabbits were anesthetized with an intramuscular injection ofketamine (35 mg/kg) and xylazine (5 mg/kg). Once deeply anesthetized,each eye was rinsed with sterile saline and IVT injections wereperformed using a 0.5 mL insulin syringe (50 units=0.5 mL) with a31-gauge needle at the supra-nasal side of the both eyes by marking theposition with a Braunstein fixed caliper (2¾″) 3.5 mm from the rectusmuscle and 4 mm from the limbus.

Tissues were collected 2 hours after the second IVT injection with IFNγ.Retina/choroid tissues (R/C) were collected and homogenized, and pSTAT1levels were measured by quantitative Western Blot on the ProteinSimpleWES instrument. The percent inhibition of IFNγ-induced pSTAT1 wascalculated in comparison to the vehicle/vehicle and vehicle/IFNγ groups.

With a 1 week pre-treatment prior to the IFNγ challenge, the suspensionformulation of compound 1 inhibited IFNγ-induced pSTAT1 by 85%. After a3 month pre-treatment with a single dose of the suspension formulationprior to the IFNγ challenge, the suspension formulation of compound 1inhibited IFNγ-induced pSTAT1 by 76%.

A suspension formulation was prepared by combining compound 1 of Example5 (Form 2), with 0.5% hydroxypropyl methylcellulose (HPMC E5)+0.02%Tween 80 in normal saline to attain a target concentration of 11.1, 3.3,and 1.1 mg/mL.

Male, New Zealand White rabbits (Liveon Biolabs, India) were used forthe studies. Animals were acclimated after arrival at the researchfacilities (Jubilant Biosys Ltd., India). Each rabbit was given a totalof two intravitreal (IVT) injections with a total dose volume of 50 μLper eye. The first IVT injection (45 μL per eye) delivered 500 μg, 150μg, or 50 μg of test compound or vehicle. Two weeks later, a second IVTinjection (5 μL per eye) delivered IFNγ (1 μg/eye; stock solution 1mg/mL; Kingfisher Biotech) or vehicle for the induction of IP-10. On theday of the injections, rabbits were anesthetized with an intramuscularinjection of ketamine (35 mg/kg) and xylazine (5 mg/kg). Once deeplyanesthetized, each eye was rinsed with sterile saline and IVT injectionswere performed using a 0.5 mL insulin syringe (50 units=0.5 mL) with a31-gauge needle at the supra-nasal side of the both eyes by marking theposition with a Braunstein fixed caliper (2¾″) 3.5 mm from the rectusmuscle and 4 mm from the limbus.

Tissues were collected 2 hours after the second IVT injection with IFNγ.Retina/choroid tissues (R/C) were collected and homogenized, and pSTAT1levels were measured by quantitative Western Blot on the ProteinSimpleWES instrument. The percent inhibition of IFNγ-induced pSTAT1 wascalculated in comparison to the vehicle/vehicle and vehicle/IFNγ groups.

With a 2 week pre-treatment prior to the IFNγ challenge, the suspensionformulation of compound 1 inhibited IFNγ-induced pSTAT1 by 79%, for the500 μg dose, by 58% for the 150 μg does and 61% for the 50 μg dose.

Assay 13: Kinome Screen and GINI Coefficient

Compounds 1 and C-1 were screened against other kinases to evaluatetheir selectivity profile.

Kinase-tagged T7 phage strains were grown in parallel in 24-well blocksin an E. coli host derived from the BL21 strain. E. coli were grown tolog-phase and infected with T7 phage from a frozen stock (multiplicityof infection=0.4) and incubated with shaking at 32° C. until lysis(90-150 minutes). The lysates were centrifuged (6,000×g) and filtered(0.2 μm) to remove cell debris. The remaining kinases were produced inHEK-293 cells and subsequently tagged with DNA for qPCR detection.

Streptavidin-coated magnetic beads were treated with biotinylated smallmolecule ligands for 30 minutes at room temperature to generate affinityresins for kinase assays. The liganded beads were blocked with excessbiotin and washed with blocking buffer (SeaBlock (Pierce), 1% BSA, 0.05%Tween 20, 1 mM DTT) to remove unbound ligand and to reduce non-specificphage binding. Binding reactions were assembled by combining kinases,liganded affinity beads, and test compounds in 1× binding buffer (20%SeaBlock, 0.17×PBS, 0.05% Tween 20, 6 mM DTT). Test compounds wereprepared as 40× stocks in 100% DMSO and directly diluted into the assay.All reactions were performed in polypropylene 384-well plates in a finalvolume of 0.04 ml. The assay plates were incubated at room temperaturewith shaking for 1 hour and the affinity beads were washed with washbuffer (1×PBS, 0.05° % Tween 20). The beads were then re-suspended inelution buffer (1×PBS, 0.05% Tween 20, 0.5 μM non-biotinylated affinityligand) and incubated at room temperature with shaking for 30 minutes.The kinase concentration in the eluates was measured by qPCR.

The compounds were screened at 1 μM, and results for primary screenbinding interactions in Table 8 and 9 are reported as “% inhibition”(=100−((test compound signal-positive control signal)/((negative controlsignal)−(positive control signal))×100) where the negative control isDMSO and the positive control is a control compound.

TABLE 8 Kinase Compound ALK AURKA CDK2 CDK7 CDK9 CSF1R EPHB6 GSK3B 1 7511 6 74 9 98 88 31 C-1 81 57 53 99 95 100 98 68

TABLE 9 Kinase Compound KIT PAK4 PKAC-ALPHA PLK4 SLK SRC SYK VEGFR2 1 8793 20 58 100 93 46 42 C-1 99 99 70 68 100 100 78 63

Compound 1 was found to exhibit significantly lower binding inhibitionfor CDK7 and CDK9 than compound C-1. Compound 1 also had lower bindinginhibition for several other kinases.

Both compounds 1 and C-1 were screened against 35 different kinases. TheGini coefficient was determined for both compounds. Compound 1 had aGINI coefficient of 0.62 and compound C-1 had a GINI coefficient of0.46. The Gini coefficient is used to express the selectivity of acompound against a panel of kinases (Graczyk, J. Med. Chem., 2007, 50,5773-5779). A higher number corresponds to a more selective compound.

The only structural difference between compound 1 and compound C-1 isthe presence of a fluoro group on the core. This structural differencehas been shown to have an important effect on the kinome selectivity ofthe compound.

Assay 14: Cytotoxicity Assay

A CellTiter-Glo luminescent cell viability/cytotoxicity assay wascarried out in BEAS-2B human lung epithelial cells (ATCC) under thenormal growth condition.

Cells were grown at 37° C. in a 5% CO₂ humidified incubator in 50%DMEM/50% F-12 medium (Life Technologies) supplemented with 10% FBS(Hyclone), 100 U/mL penicillin, 100 μg/mL streptomycin (LifeTechnologies), and 2 mM GlutaMAX (Life Technologies). On day 1 of theassay, cells were seeded at a 500 cells/well density in white 384-welltissue culture plates (Corning) with 25 μL medium, and were allowed toadhere overnight in the incubator. On day 2 of the assay, 5 μL of mediumcontaining dose-responses of test compounds was added, and incubated at37° C. for 48 h. 30 μL of CellTiter-Glo detection solution (Promega) wassubsequently added, mixed on an orbital shaker for 5 min, and incubatedfor additional 10 min before being read on the EnVision reader.Luminescence signals were recorded and percent DMSO control values werecalculated.

For dose-response analysis, percent DMSO control data were plotted vs.compound concentrations to derive dose-response curves by lineconnecting each data point. The concentration at which each curvecrosses the 15% inhibition threshold is defined as CC₁₅.

It is expected that test compounds exhibiting a higher CC₁₅ value inthis assay have less likelihood to cause cytotoxicity.

Compound 1 exhibited a CC₁₅ of 3.16 μM whereas compound C-1 exhibited aCC₁₅ of 630 nM. Therefore, compound 1 is significantly less likely tocause cytotoxicity than compound C-1 based on this assay.

The only structural difference between compound 1 and compound C-1 isthe presence of a fluoro group on the core. This structural differencehas been shown to have an important effect on the cytotoxicity of thecompound.

While the present invention has been described with reference tospecific aspects or embodiments thereof, it will be understood by thoseof ordinary skilled in the art that various changes can be made orequivalents can be substituted without departing from the true spiritand scope of the invention. Additionally, to the extent permitted byapplicable patent statutes and regulations, all publications, patentsand patent applications cited herein are hereby incorporated byreference in their entirety to the same extent as if each document hadbeen individually incorporated by reference herein.

1-23. (canceled)
 24. A method of treating an ocular disease in a mammal,the method comprising administering a pharmaceutical compositioncomprising a compound of formula:

or a pharmaceutically-acceptable salt thereof, and apharmaceutically-acceptable carrier to the eye of the mammal.
 25. Themethod of claim 24, wherein the ocular disease is uveitis, diabeticretinopathy, diabetic macular edema, dry eye disease, age-relatedmacular degeneration, retinal vein occlusion, or atopickeratoconjunctivitis.
 26. The method of claim 25 wherein the oculardisease is uveitis. 27-31. (canceled)
 32. The method of claim 25,wherein the ocular disease is diabetic macular edema.
 33. The method ofclaim 24, wherein the pharmaceutical composition is administered byintravitreal injection.
 34. The method of claim 33, wherein thepharmaceutical composition is a suspension.
 35. A method of treating anocular disease in a mammal, the method comprising administering to theeye of the mammal a pharmaceutical composition comprising a crystallineform of the compound of formula:

wherein the crystalline form is characterized by a powder X-raydiffraction pattern comprising diffraction peaks at 2θ values of10.61±0.20, 11.84±0.20, 14.94±0.20, 18.26±0.20, and 19.06±0.20, and apharmaceutically-acceptable carrier.
 36. The method of claim 35, whereinthe powder X-ray diffraction pattern is further characterized by havingadditional diffraction peaks at 2θ values of 13.32±0.20, 17.69±0.20, and21.10±0.20.
 37. The method of claim 35, wherein the powder X-raydiffraction pattern is further characterized by having two or moreadditional diffraction peaks at 2θ values selected from 10.85±0.20,16.14±0.20, 16.35±0.20, 18.43±0.20, 19.20±0.20, 19.49±0.20, 20.72±0.20,21.94±0.20, 22.64±0.20, 23.64±0.20, 25.19±0.20, and 28.08±0.20.
 38. Themethod of claim 35, wherein the crystalline form is characterized by apowder X-ray diffraction pattern in which the peak positions aresubstantially in accordance with the peak positions of the pattern shownin FIG.
 1. 39. The method of claim 35, wherein the crystalline form ischaracterized by a differential scanning calorimetry trace recorded at aheating rate of 10° C. per minute which shows a maximum in endothermicheat flow at a temperature between 268° C. and 277° C.
 40. The method ofclaim 35, wherein the crystalline form is characterized by adifferential scanning calorimetry trace substantially in accordance withthat shown in FIG.
 2. 41. The method of claim 35, wherein the oculardisease is uveitis, diabetic retinopathy, diabetic macular edema, dryeye disease, age-related macular degeneration, retinal vein occlusion,or atopic keratoconjunctivitis.
 42. The method of claim 41 wherein theocular disease is uveitis.
 43. The method of claim 41 wherein the oculardisease is diabetic macular edema.
 44. The method of claim 35, whereinthe pharmaceutical composition is administered by intravitrealinjection.
 45. The method of claim 44, wherein the pharmaceuticalcomposition is a suspension.
 46. A method of treating an ocular diseasein a mammal, the method comprising administering to the eye of themammal a pharmaceutical composition comprising a crystalline form of thecompound of formula:

wherein the crystalline form is characterized by a powder X-raydiffraction pattern comprising diffraction peaks at 2θ values of8.16±0.20, 8.97±0.20, 15.29±0.20, 16.70±0.20, 18.00±0.20, and20.18±0.20, and a pharmaceutically-acceptable carrier.
 47. The method ofclaim 46, wherein the powder X-ray diffraction pattern is furthercharacterized by having two or more additional diffraction peaks at 2θvalues selected from 7.69±0.20, 10.66±0.20, 11.46±0.20, 11.91±0.20,15.80±0.20, 17.02±0.20, 18.83±0.20, 22.39±0.20, 22.98±0.20, 24.89±0.20,and 26.54±0.20.
 48. The method of claim 46, wherein the crystalline formis characterized by a powder X-ray diffraction pattern in which the peakpositions are substantially in accordance with the peak positions of thepattern shown in FIG.
 6. 49. The method of claim 46, wherein thecrystalline form is characterized by a differential scanning calorimetrytrace recorded at a heating rate of 10° C. per minute which a maximum inendothermic heat flow at a temperature between 215° C. and 229° C. 50.The method of claim 46, wherein the crystalline form is characterized bya differential scanning calorimetry trace substantially in accordancewith that shown in FIG.
 7. 51. The method of claim 46, wherein theocular disease is uveitis, diabetic retinopathy, diabetic macular edema,dry eye disease, age-related macular degeneration, retinal veinocclusion, or atopic keratoconjunctivitis.
 52. The method of claim 51wherein the ocular disease is uveitis.
 53. The method of claim 51wherein the ocular disease is diabetic macular edema.
 54. The method ofclaim 46, wherein the pharmaceutical composition is administered byintravitreal injection.
 55. The method of claim 54, wherein thepharmaceutical composition is a suspension.